Loss of BMAL1 in ovarian steroidogenic cells results in implantation failure in female mice.

The circadian clock plays a significant role in many aspects of female reproductive biology, including estrous cycling, ovulation, embryonic implantation, onset of puberty, and parturition. In an effort to link cell-specific circadian clocks to their specific roles in female reproduction, we used the promoter that controls expression of Steroidogenic Factor-1 (SF1) to drive Cre-recombinase-mediated deletion of the brain muscle arnt-like 1 (Bmal1) gene, known to encode an essential component of the circadian clock (SF1-Bmal1(-/-)). The resultant SF1-Bmal1(-/-) females display embryonic implantation failure, which is rescued by progesterone supplementation, or bilateral or unilateral transplantation of wild-type ovaries into SF1-Bmal1(-/-) dams. The observation that the central clock, and many other peripheral clocks, are fully functional in this model allows the assignment of the implantation phenotype to the clock in ovarian steroidogenic cells and distinguishes it from more general circadian related systemic pathology (e.g., early onset arthropathy, premature aging, ovulation, late onset of puberty, and abnormal estrous cycle). Our ovarian transcriptome analysis reveals that deletion of ovarian Bmal1 disrupts expression of transcripts associated with the circadian machinery and also genes critical for regulation of progesterone production, such as steroidogenic acute regulatory factor (Star). Overall, these data provide a powerful model to probe the interlocking and synergistic network of the circadian clock and reproductive systems.

Hepatocyte circadian clock controls acetaminophen bioactivation through NADPH-cytochrome P450 oxidoreductase.

The diurnal variation in acetaminophen (APAP) hepatotoxicity (chronotoxicity) reportedly is driven by oscillations in metabolism that are influenced by the circadian phases of feeding and fasting. To determine the relative contributions of the central clock and the hepatocyte circadian clock in modulating the chronotoxicity of APAP, we used a conditional null allele of brain and muscle Arnt-like 1 (Bmal1, aka Mop3 or Arntl) allowing deletion of the clock from hepatocytes while keeping the central and other peripheral clocks (e.g., the clocks controlling food intake) intact. We show that deletion of the hepatocyte clock dramatically reduces APAP bioactivation and toxicity in vivo and in vitro because of a reduction in NADPH-cytochrome P450 oxidoreductase gene expression, protein, and activity.

Familial isolated pituitary adenoma is independent of Ahr genotype in a novel mouse model of disease.

Human familial isolated pituitary adenoma (FIPA) has been linked to germline heterozygous mutations in the gene encoding the aryl hydrocarbon receptor-interacting protein (AIP, also known as ARA9, XAP2, FKBP16, or FKBP37). To investigate the hypothesis that AIP is a pituitary adenoma tumor suppressor via its role in aryl hydrocarbon receptor (AHR) signaling, we have compared the pituitary phenotype of our global null Aip (AipΔC) mouse model with that of a conditional null Aip model (Aipfx/fx) carrying the same deletion, as well as pituitary phenotypes of Ahr global null and Arnt conditional null animals. We demonstrate that germline AipΔC heterozygosity results in a high incidence of pituitary tumors in both sexes, primarily somatotropinomas, at 16 months of age. Biallelic deletion of Aip in Pit-1 cells (Aipfx/fx:rGHRHRcre) increased pituitary tumor incidence and also accelerated tumor progression, supporting a loss-of-function/loss-of-heterozygosity model of tumorigenesis. Tumor development exhibited sexual dimorphism in wildtype and Aipfx/fx:rGHRHRcre animals. Despite the role of AHR as a tumor suppressor in other cancers, the observation that animals lacking AHR in all tissues, or ARNT in Pit-1 cells, do not develop somatotropinomas argues against the hypothesis that pituitary tumorigenesis in AIP-associated FIPA is related to decreased activities of either the Ahr or Arnt gene products.

The aryl hydrocarbon receptor-interacting protein (AIP) is required for dioxin-induced hepatotoxicity but not for the induction of the Cyp1a1 and Cyp1a2 genes.

The aryl hydrocarbon receptor (AHR) plays an essential role in the toxic response to environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), in the adaptive up-regulation of xenobiotic metabolizing enzymes, and in hepatic vascular development. In our model of AHR signaling, the receptor is found in a cytosolic complex with a number of molecular chaperones, including Hsp90, p23, and the aryl hydrocarbon receptor-interacting protein (AIP), also known as ARA9 and XAP2. To understand the role of AIP in adaptive and toxic aspects of AHR signaling, we generated a conditional mouse model where the Aip locus can be deleted in hepatocytes. Using this model, we demonstrate two important roles for the AIP protein in AHR biology. (i) The expression of AIP in hepatocytes is essential to maintain high levels of functional cytosolic AHR protein in the mammalian liver. (ii) Expression of the AIP protein is essential for dioxin-induced hepatotoxicity. Interestingly, classical AHR-driven genes show differential dependence on AIP expression. The Cyp1b1 and Ahrr genes require AIP expression for normal up-regulation by dioxin, whereas Cyp1a1 and Cyp1a2 do not. This differential dependence on AIP provides evidence that the mammalian genome contains more than one class of AHR-responsive genes and suggests that a search for AIP-dependent, AHR-responsive genes may guide us to the targets of the dioxin-induced hepatotoxicity.

Generation of an Allelic Series at the Ahr Locus Using an Edited Recombinant Approach.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and a member of the PER-ARNT-SIM (PAS) superfamily of environmental sensors. The AHR is involved in a series of biological processes including adaptive metabolism of xenobiotics, toxicity of certain environmental pollutants, vascular development, fertility, and immune function. Mouse models, including the Ahr null and Ahr conditional null (Ahrfx) mice, are widely used for the study of AHR-mediated biology and toxicity. The Ahr conditional null mouse harbors the low-affinity Ahrd allele that exhibits approximately a 10-fold lower binding affinity for certain xenobiotic AHR ligands than the widely used C57BL/6 mouse that harbors the higher affinity Ahrb1 allele. Here, we report a novel mouse model that introduces a V375A polymorphism that converts the low-affinity allele into a high-affinity allele, offering a more sensitive conditional model. In the generation of this novel conditional allele, two additional mutants arose, including a 3-bp deletion in the PAS-B domain (AhrNG367R) and an early termination codon in the PAS-B domain (AhrTer383). The AhrNG367R allele presents as a phenocopy of the null and the AhrTer383 allele presents as an antimorph when assessing for the ductus venosus and liver lobe weight endpoints. These new models represent a series of tools that will be useful in further characterizing AHR biology.

Who participates in state sponsored Medicaid enhanced prenatal services?

Medicaid insures an estimated 43% of all births in Michigan and provides additional funding for enhanced prenatal services (EPS). The objectives of this study are to report on the (1) use of statewide administrative data to examine risk characteristics and EPS enrollment of Medicaid-insured pregnant women in Michigan; and (2) presence and extent of a broad range of risk factors in a sample of EPS participants in Michigan, using a newly developed two-tier, risk screener and assessment tool. This study uses Vital Records, Medicaid and other data to describe EPS participation by maternal risks in the statewide population of Medicaid-insured pregnant women (54,582 in the fiscal year 2005). The screener study data is a convenience sample of 2,203 women screened between February 2005 and October 2007. The administrative data indicates that 26% of Medicaid-eligible pregnant women had EPS contact. Most women with health behavior risks, such as smoking and drug use, had no contact with EPS (68-72%). Approximately 58% of all Medicaid-insured women had zero to two co-occurring risks, while 42% had three or more of the analyzed risks. Among screened women who smoke, 9% smoked more than a pack a day. Approximately 34% of women with a depression screen scored in the moderately or severely depressed range. The results of this study suggest great opportunity for EPS enhancement by improving the capacity to identify and engage women with modifiable risks, match interventions to specific health problems, and deliver services at an intensity warranted by the risk level.

Retinal pathology in the PPCD1 mouse.

Retinal phenotypes of the PPCD1 mouse, a mouse model of posterior polymorphous corneal dystrophy, have been characterized. PPCD1 mice on the DBA/2J background (D2.Ppcd1) have previously been reported to develop an enlarged anterior chamber due to epithelialization and proliferation of the corneal endothelium and subsequent blockage of the iridocorneal angle. Results presented here show that D2.Ppcd1 mice develop increased intraocular pressure (IOP), with measurements at three months of age revealing significant increases in IOP. Significant retinal ganglion cell layer cell loss is observed at five months of age. D2.Ppcd1 animals also exhibit marked degeneration of the outer nuclear layer in association with hyperplasia of the retinal pigment epithelium. Evidence of retinal detachment is present as early as three weeks of age. By 3.5 months of age, focal areas of outer nuclear layer loss are observed. Although the GpnmbR150X mutation leads to increased IOP and glaucoma in DBA/2J mice, development of anterior segment and retinal defects in D2.Ppcd1 animals does not depend upon presence of the GpnmbR150X mutation.

Liver tumor promotion by 2,3,7,8-tetrachlorodibenzo-p-dioxin is dependent on the aryl hydrocarbon receptor and TNF/IL-1 receptors.

We set out to better understand the signal transduction pathways that mediate liver tumor promotion by 2,3,7,8-tetrachlorodibenzo-p-dioxn ("dioxin"). To this end, we first employed congenic mice homozygous for either the Ahr(b1) or Ahr(d) alleles (encoding an aryl hydrocarbon receptor (AHR) with high or low binding affinity for dioxin, respectively) and demonstrated that hepatocellular tumor promotion in response to dioxin segregated with the Ahr locus. Once we had genetic evidence for the importance of AHR signaling, we then asked if tumor promotion by dioxin was influenced by "interleukin-1 (IL-1)-like" inflammatory cytokines. The importance of this question arose from our earlier observation that aspects of the acute hepatocellular toxicity of dioxin are dependent upon IL1-like cytokine signaling. To address this issue, we employed a triple knock-out (TKO) mouse model with null alleles at the loci encoding the three relevant receptors for tumor necrosis factors α and ß and IL-1α and IL-1ß (i.e., null alleles at the Tnfrsf1a, Tnfrsf1b, and Il-1r1 loci). The observation that TKO mice were resistant to the tumor promoting effects of dioxin in liver suggests that inflammatory cytokines play an important step in dioxin mediated liver tumor promotion in the mouse. Collectively, these data support the idea that the mechanism of dioxin acute hepatotoxicity and its activity as a promoter in a mouse two stage liver cancer model may be similar, i.e., tumor promotion by dioxin, like acute hepatotoxicity, are mediated by the linked action of two receptor systems, the AHR and the receptors for the "IL-1-like" cytokines.

Aryl hydrocarbon receptor nuclear translocator in hepatocytes is required for aryl hydrocarbon receptor-mediated adaptive and toxic responses in liver.

The aryl hydrocarbon receptor (AHR) plays a central role in the toxic responses to halogenated dibenzo-p-dioxins ("dioxins"), in the metabolic adaptation to polycyclic aromatic hydrocarbons, and in the development of the mature vascular system. A number of lines of evidence support the idea that the regulation of adaptive metabolism requires an AHR partnership with the aryl hydrocarbon receptor nuclear translocator (ARNT). Yet, for AHR-dependent vascular development and dioxin toxicity, the role of ARNT is less certain. In fact, numerous models have been proposed over the years to suggest that the AHR signals in important ways via ARNT-independent events. In an effort to clarify the role of ARNT in AHR-mediated dioxin hepatotoxicity, we generated a conditional Arnt mouse model. Such a model was essential because global inactivation of Arnt results in embryonic lethality presumably due to this protein's role as a heterodimeric partner for the hypoxia-inducible factors (HIFs). Using a hepatocyte-specific Arnt deletion, we were able to demonstrate that hepatocyte ARNT is required for major aspects of AHR-mediated dioxin toxicity in the liver. Results from this conditional Arnt allele are also consistent with a model where hepatocyte ARNT is unrelated to AHR-mediated hepatovascular development. In sum, these data suggest that AHR-ARNT dimers within the hepatocyte direct the toxic and adaptive and developmental functions associated with the AHR and that developmental vascular events arise due to signaling in a distinct cell type expressing this dimeric pair.

Abnormal liver development and resistance to 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in mice carrying a mutation in the DNA-binding domain of the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) is known for its role in the adaptive and toxic responses to a large number of environmental contaminants, as well as its role in hepatovascular development. The classical AHR pathway involves ligand binding, nuclear translocation, heterodimerization with the AHR nuclear translocator (ARNT), and binding of the heterodimer to dioxin response elements (DREs), thereby modulating the transcription of an array of genes. The AHR has also been implicated in signaling events independent of nuclear localization and DNA binding, and it has been suggested that such pathways may play important roles in the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Here, we report the generation of a mouse model that expresses an AHR protein capable of ligand binding, interactions with chaperone proteins, functional heterodimerization with ARNT, and nuclear translocation, but is unable to bind DREs. Using this model, we provide evidence that DNA binding is required AHR-mediated liver development, as Ahr(dbd/dbd) mice exhibit a patent ductus venosus, similar to what is seen in Ahr(-/-) mice. Furthermore, Ahr(dbd/dbd) mice are resistant to TCDD-induced toxicity for all endpoints tested. These data suggest that DNA binding is necessary for AHR-mediated developmental and toxic signaling.

Hepatic transcriptional networks induced by exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) serves as a prototype for a range of environmental toxicants and as a pharmacologic probe to study signal transduction by the aryl hydrocarbon receptor (AHR). Despite a detailed understanding of how TCDD exposure leads to the transcriptional up-regulation of cytochrome P450-dependent monooxygenases, we know little about how compounds like TCDD lead to a variety of AHR-dependent toxic end points such as liver pathology, terata, thymic involution, and cancer. Using an acute exposure protocol and the toxic response of the mouse liver as a model system, we have begun a detailed microarray analysis to describe the transcriptional changes that occur after various TCDD doses and treatment times. Through correlation analysis of time- and dose-dependent toxicological end points, we are able to identify coordinately responsive transcriptional events that can be defined as primary transcriptional events and downstream events that may represent mechanistically linked sequelae or that have potential as biomarkers of toxicity.

Aspects of dioxin toxicity are mediated by interleukin 1-like cytokines.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) results in a broad spectrum of toxic effects. Most, if not all, of these responses are dependent upon the binding of dioxin to the aryl hydrocarbon receptor. Given their common roles in chemically induced toxicity, we asked whether interleukin 1 (IL1)-like cytokines play a role in acute aspects of the dioxin response. To test this idea, we employed a "triple-null" mouse model that lacks the two receptors for the tumor necrosis factors-alpha and -beta and the receptor for the IL1-alpha and IL1-beta cytokines. When triple null mice were treated with dioxin, there was significant attenuation in the levels of serum alanine aminotransferase, signifying reduced hepatocellular damage. In addition, the triple-null mice were protected from dioxin-induced liver inflammation. Loss of receptors for the IL1-like cytokines was not protective for all aspects of dioxin toxicity. Endpoints such as thymic involution, Cyp1a2 induction, hepatomegaly, and hydropic degeneration remain unchanged in this model.

Progressive arthropathy in mice with a targeted disruption of the Mop3/Bmal-1 locus.

Disruption of the murine Mop3 (also known as Bmal1 or Arntl) locus results in a loss of behavioral and molecular circadian rhythms. Although Mop3 null mice do not display anomalies in early development, they do display reduced activity as they age. In an effort to explain this decreased activity, we characterized the physiological and anatomical changes that occurred with age. We observed that Mop3 null mice display an increased mortality after 26 weeks of age and a phenotype best described as a progressive noninflammatory arthropathy. Although little pathology is observed prior to 11 weeks of age, by 35 weeks of age essentially all Mop3 null animals develop joint ankylosis due to flowing ossification of ligaments and tendons and almost complete immobilization of weight-bearing and nonweight-bearing joints. This pathology appears to explain the decreased activity of Mop3 null mice and suggests that MOP3 is an inhibitor of ligament and tendon ossification.

Resistance to 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity and abnormal liver development in mice carrying a mutation in the nuclear localization sequence of the aryl hydrocarbon receptor.

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.