Optimized J to T peak and T peak to T end measurements in nonclinical species administered moxifloxacin and amiodarone.

INTRODUCTION: Cardiovascular safety and the risk of developing the potentially fatal ventricular tachyarrhythmia, Torsades de Pointes (TdP), have long been major concerns of drug development. TdP is associated with a delayed ventricular repolarization represented by QT interval prolongation in the electrocardiogram (ECG), typically due to block of the potassium channel encoded by the human ether-a-go-go related gene (hERG). Importantly however, not all drugs that prolong the QT interval are torsadagenic and not all hERG blockers prolong the QT interval. Recent clinical reports suggest that partitioning the QT interval into early (J to T peak; JTp) and late repolarization (T peak to T end; TpTe) components may be valuable for distinguishing low-risk mixed ion channel blockers (hERG plus calcium and/or late sodium currents) from high-risk pure hERG channel blockers. This strategy, if true for nonclinical animal models, could be used to de-risk QT prolonging compounds earlier in the drug development process. METHODS: To explore this, we investigated JTp and TpTe in ECG data collected from telemetered dogs and/or monkeys administered moxifloxacin or amiodarone at doses targeting relevant clinical exposures. An optimized placement of the Tpeak fiducial mark was utilized, and all intervals were corrected for heart rate (QTc, JTpc, TpTec). RESULTS: Increases in QTc and JTpc intervals with administration of the pure hERG blocker moxifloxacin and an initial QTc and JTpc shortening followed by prolongation with the mixed ion channel blocker amiodarone were detected as expected, aligning with clinical data. However, anticipated increases in TpTec by both standard agents were not detected. DISCUSSION: The inability to detect changes in TpTec reduces the utility of these subintervals for prediction of arrhythmias using continuous single­lead ECGs collected from freely moving dogs and monkeys.

Differential effects of light and feeding on circadian organization of peripheral clocks in a forebrain Bmal1 mutant.

In order to assess the contribution of a central clock in the hypothalamic suprachiasmatic nucleus (SCN) to circadian behavior and the organization of peripheral clocks, we generated forebrain/SCN-specific Bmal1 knockout mice by using floxed Bmal1 and pan-neuronal Cre lines. The forebrain knockout mice showed >90% deletion of BMAL1 in the SCN and exhibited an immediate and complete loss of circadian behavior in constant conditions. Circadian rhythms in peripheral tissues persisted but became desynchronized and damped in constant darkness. The loss of synchrony was rescued by light/dark cycles and partially by restricted feeding (only in the liver and kidney but not in the other tissues) in a distinct manner. These results suggest that the forebrain/SCN is essential for internal temporal order of robust circadian programs in peripheral clocks, and that individual peripheral clocks are affected differently by light and feeding in the absence of a functional oscillator in the forebrain.

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.

Effects of lersivirine on canine and rodent thyroid function.

Lersivirine is a nonnucleoside reverse transcriptase inhibitor (NNRTI) being developed for the treatment of HIV-1 infection. Like other NNRTIs, lersivirine is a potent enzyme inducer in rodents capable of inducing a number of hepatic enzymes including those involved in its own metabolism. Preclinically lersivirine has been associated with hepatocellular hypertrophy and thyroid gland follicular cell hypertrophy in rats, mice, and dogs. In rodents, we show that development of thyroid hypertrophy is related to the classic mechanism, namely increased thyroxine (T4) clearance secondary to induction of uridine-diphosphoglucuronosyltransferase (UDPGT) in the liver and a resulting increase in thyroid-stimulating hormone. Similarly, lersivirine-exposed dogs exhibit a significant increase in hepatic UDPGT enzyme activity along with increased T4 clearance although clear effects on serum thyroid hormone levels were less apparent. These effects on thyroid hormonal clearance in the dog suggest that thyroid gland hypertrophy in this species is due to the same mechanism shown to occur in rodents although, as expected, dogs better adapt to these effects and therefore maintain relatively normal thyroid hormonal balance. It is also notable that the minimal thyroid follicular hypertrophy that occurs in dogs does not progress as is seen in rodents. As is the case with rodents, these adaptive changes in the dog are not considered indicative of a human health risk.

Intravenous solid tip lead placement in telemetry implanted dogs. Part 1: Surgical methods, signal quality, and pathological endpoints.

INTRODUCTION: Electrocardiogram (ECG) signals in safety pharmacology studies are generally collected via subcutaneous or epicardial leads. Subcutaneous placement is an easier procedure, but signals often contain artifacts. Epicardial leads offer improved quality but require additional surgical expertise. Signal quality and tolerability of intravenous (IV)/diaphragmatic ECG leads were investigated as a less invasive alternative to the epicardial ECG lead approach for cardiovascular assessment in dogs. METHODS: Twenty-eight beagle dogs were implanted with PCT (n=14) or PCTP (n=14) transmitters with IV (negative)/diaphragmatic (positive) ECG leads arranged in approximate Lead II configuration. Surgical time for previous epicardial and current IV lead placement approaches was compared. The ECG signals were assessed for up to 32 weeks post-surgery. Signal quality was assessed based on good wave/total wave (GW/TW) ratios calculated using ECG PRO (Ponemah Physiology Platform, Version 4.8) and variability in ECG parameter measurements for each surgical model. Clinical pathology was assessed on all animals before surgery and approximately 2 and 12 weeks post-surgery. A specialized necropsy was conducted on four animals (two PCT and two PCTP) to assess the tolerability of telemetry equipment; selected tissues were examined microscopically. RESULTS: Surgical time using the IV lead method was approximately 18% shorter than the epicardial lead method. The GW/TW ratio for IV lead-implanted dogs indicated good durability of signal that was similar to epicardial leads. Intra- and inter-animal variability in ECG parameter measurements was similar between IV lead-implanted and epicardial lead-implanted dogs. Clinical pathology revealed no noteworthy findings, and the IV/diaphragmatic surgical approach had minimal consequences on local vasculature and associated implantation sites. DISCUSSION: Advantages of the IV/diaphragmatic lead model include a less invasive and shorter surgical procedure; high tissue tolerance, ECG signal quality, and durability; and data processing procedures similar to that of epicardial leads. Therefore, the IV/diaphragmatic lead configuration is a viable alternative to more invasive surgical approaches for telemetry device implantation in dogs.

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.

A hypomorphic allele of aryl hydrocarbon receptor-associated protein-9 produces a phenocopy of the AHR-null mouse.

The aryl hydrocarbon receptor-associated protein-9 (ARA9) is a chaperone of the aryl hydrocarbon receptor (AHR). The AHR has been shown to play a late developmental role in the normal closure of a fetal hepatovascular shunt known as the ductus venosus (DV). Given that Ara9-null mice display early embryonic lethality, we generated a hypomorphic Ara9 allele (designated Ara9(fxneo)) that displays reduced ARA9 protein expression. In an effort to demonstrate the role of ARA9 protein in AHR-mediated DV closure, we used combinations of Ara9 wild-type [Ara9(+/+)], null [Ara9(-/-)], and hypomorphic [Ara9(fxneo/fxneo)] alleles to produce mice with a graded expression of the ARA9 protein. Liver perfusion studies demonstrated that although none of the Ara9(+/+) mice displayed a patent DV, the shunt was observed in 10% of the Ara9(+/fxneo) mice, 55% of the Ara9(+/-) mice, and 83% of the Ara9(fxneo/fxneo) mice. That expression level of ARA9 correlates with the frequency of a phenocopy of the Ahr-null allele supports the conclusion that the ARA9 protein is essential for AHR signaling during development.

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.

Genetic components of the circadian clock regulate thrombogenesis in vivo.

BACKGROUND: Myocardial infarction, stroke, and sudden death undergo diurnal variation. Although genes relevant to hemostasis and vascular integrity undergo circadian oscillation, the role of the molecular clock in thrombotic events remains to be established. METHODS AND RESULTS: A diurnal variation in the time to thrombotic vascular occlusion (TTVO) subsequent to a photochemical injury was observed in wild-type mice: TTVO varied from 24.6+/-2.7 minutes at zeitgeber time (ZT) 2 to 40.3+/-4.3 minutes at ZT8, 24.3+/-2.3 minutes at ZT14, and 31.0+/-4.4 minutes at ZT20. This pattern was disrupted or altered when core clock genes-BMAL1, CLOCK, and NPAS2-were mutated or deleted. Mutation of CLOCK abolished the diurnal variation in TTVO, whereas deletion of NPAS2 altered its temporal pattern. NPAS2 deletion prolonged TTVO and reduced blood pressure irrespective of clock time. Global BMAL1 deletion shortened TTVO at ZT8, and the diurnal variation in TTVO, but not in systemic blood pressure, was disrupted in mice in which BMAL1 had been selectively deleted in endothelium. CONCLUSIONS: Key components of the molecular clock regulate the response to a thrombogenic stimulus in vivo. Such a phenomenon may interact with environmental variables, and together with the influence of these genes on blood pressure may contribute to the diurnal variation in cardiovascular events observed in humans.

Dissecting the functions of the mammalian clock protein BMAL1 by tissue-specific rescue in mice.

The basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) domain transcription factor BMAL1 is an essential component of the mammalian circadian pacemaker. Bmal1-/- mice lose circadian rhythmicity but also display tendon calcification and decreased activity, body weight, and longevity. To investigate whether these diverse functions of BMAL1 are tissue-specific, we produced transgenic mice that constitutively express Bmal1 in brain or muscle and examined the effects of rescued gene expression in Bmal1-/- mice. Circadian rhythms of wheel-running activity were restored in brain-rescued Bmal1-/- mice in a conditional manner; however, activity levels and body weight were lower than those of wild-type mice. In contrast, muscle-rescued Bmal1-/- mice exhibited normal activity levels and body weight yet remained behaviorally arrhythmic. Thus, Bmal1 has distinct tissue-specific functions that regulate integrative physiology.

A time to divide: does the circadian clock control cell cycle?

A variety of molecular, genomic and epidemiological evidence has linked cell cycle regulation to circadian rhythms. In a recent issue of Molecular Cell, Koeffler and colleagues show that PER1 sensitizes human cancer cells to ionizing radiation-induced apoptosis. In addition, PER1 expression was found to be downregulated in human tumors, suggesting a tumor suppressor role for the PER1 protein. The significance of PERs, clocks, and cell cycle control is discussed.

Aryl hydrocarbon receptor-dependent liver development and hepatotoxicity are mediated by different cell types.

The aryl hydrocarbon receptor (AHR) plays a role in three areas of biology that include the adaptive metabolism of xenobiotics, the toxic responses associated with exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), and vascular remodeling of the developing embryo. To test the hypothesis that receptor signaling in different cell types is responsible for these aspects of AHR biology, we generated a conditional Ahr allele where exon 2 is flanked by loxP sites. Through the use of Cre-lox technology, we then investigated the role of AHR signaling in hepatocytes or endothelial cells in mediating prototypical endpoints of adaptive, toxic, or developmental signaling. Using this model, we provide evidence that AHR signaling in endothelial/hematopoietic cells is necessary for developmental closure of the ductus venosus, whereas AHR signaling in hepatocytes is necessary to generate adaptive and toxic responses of the liver in response to dioxin exposure. Taken together, these data illustrate the importance of cell-specific receptor signaling for the generation of distinct AHR-dependent physiological outcomes.

Inactivation of the arylhydrocarbon receptor nuclear translocator (Arnt) suppresses von Hippel-Lindau disease-associated vascular tumors in mice.

Patients with germ line mutations in the VHL tumor suppressor gene are predisposed to the development of highly vascularized tumors within multiple tissues. Loss of pVHL results in constitutive activation of the transcription factors HIF-1 and HIF-2, whose relative contributions to the pathogenesis of the VHL phenotype have yet to be defined. In order to examine the role of HIF in von Hippel-Lindau (VHL)-associated vascular tumorigenesis, we utilized Cre-loxP-mediated recombination to inactivate hypoxia-inducible factor-1alpha (Hif-1alpha) and arylhydrocarbon receptor nuclear translocator (Arnt) genes in a VHL mouse model of cavernous liver hemangiomas and polycythemia. Deletion of Hif-1alpha did not affect the development of vascular tumors and polycythemia, nor did it suppress the increased expression of vascular endothelial growth factor (Vegf) and erythropoietin (Epo). In contrast, phosphoglycerokinase (Pgk) expression was substantially decreased, providing evidence for target gene-dependent functional redundancy between different Hif transcription factors. Inactivation of Arnt completely suppressed the development of hemangiomas, polycythemia, and Hif-induced gene expression. Here, we demonstrate genetically that the development of VHL-associated vascular tumors in the liver depends on functional ARNT. Furthermore, we provide evidence that individual HIF transcription factors may play distinct roles in the development of specific VHL disease manifestations.

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.

EDGE: a centralized resource for the comparison, analysis, and distribution of toxicogenomic information.

Transcriptional profiling via microarrays holds great promise for toxicant classification and hazard prediction. Unfortunately, the use of different microarray platforms, protocols, and informatics often hinders the meaningful comparison of transcriptional profiling data across laboratories. One solution to this problem is to provide a low-cost and centralized resource that enables researchers to share toxicogenomic data that has been generated on a common platform. In an effort to create such a resource, we developed a standardized set of microarray reagents and reproducible protocols to simplify the analysis of liver gene expression in the mouse model. This resource, referred to as EDGE, was then used to generate a training set of 117 publicly accessible transcriptional profiles that can be accessed at http://edge.oncology.wisc.edu/. The Web-accessible database was also linked to an informatics suite that allows on-line clustering and K-means analyses as well as Boolean and sequence-based searches of the data. We propose that EDGE can serve as a prototype resource for the sharing of toxicogenomics information and be used to develop algorithms for efficient chemical classification and hazard prediction.

Gestational exposure of Ahr and Arnt hypomorphs to dioxin rescues vascular development.

The aryl hydrocarbon receptor (AHR) is commonly known for its role in the adaptive metabolism of xenobiotics and in the toxic events that follow exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin). Previously, we have demonstrated that the AHR and its heterodimeric partner, the AHR nuclear translocator (ARNT), play a role in the developmental closure of a hepatic vascular shunt known as the ductus venosus (DV). To investigate the mechanism of DV closure, we generated hypomorphic alleles of the Ahr and Arnt loci. Using these models, we then asked whether this vascular defect could be rescued by receptor activation during late development. By manipulating gestational exposure, the patent DV in AHR or ARNT hypomorphs could be efficiently closed by dioxin exposure as early as embryonic day 12.5 and as late as embryonic day 18.5. These findings define the temporal regulation of receptor activation during normal ontogeny and provide evidence to support the idea that receptor activation and AHR-ARNT heterodimerization are essential for normal vascular development. Taken in the broader context, these data demonstrate that similar AHR signaling steps govern all major aspects of AHR biology.

Patent ductus venosus and dioxin resistance in mice harboring a hypomorphic Arnt allele.

The Ah receptor nuclear translocator (ARNT) is the dimeric partner of hypoxia-inducible factors and thus plays a pivotal role in cellular adaptation to low oxygen environments. ARNT is also a dimeric partner for the Ah receptor (AHR), and this complex is essential in regulating the adaptive metabolic response to polycyclic aromatic hydrocarbons. Because of the essential role of ARNT in hypoxia-driven developmental events, it has been difficult to study the physiological significance of AHR.ARNT heterodimers in vivo. To address this issue, we developed a hypomorphic Arnt allele that displayed normal development and allowed the examination of the role of ARNT in AHR biology. In this regard, the AHR is also known to mediate two additional biological processes: the toxicological response to compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) and the developmental closure of a fetal vascular structure known as the ductus venosus. Although the mechanism of the adaptive pathway has been well described, the mechanism of AHR-mediated signal transduction in the toxic and developmental pathways is not well understood. Liver perfusion studies demonstrated that ARNT hypomorphs have a patent ductus venosus, identical to that observed in the Ahr null mice. Parallel dioxin toxicity studies demonstrated that the ARNT hypomorphs exhibited resistance to the end points of dioxin exposure. Moreover, we observed that toxicity could be segregated from the classical adaptive responses such as P4501A induction. Taken in sum, these experiments demonstrate that ARNT is an essential component of AHR developmental signaling and shed light on the mechanism of dioxin toxicity.