Whole exome and transcript profiling of liver following aflatoxin B1 exposure in rats.

We recently developed a rat whole exome sequencing (WES) panel and used it to evaluate early somatic mutations in archival liver tissues from F344/N rats exposed to the hepatocarcinogen, Aflatoxin B1 (AFB1), a widely studied, potent mutagen and hepatocarcinogen associated with hepatocellular carcinoma (HCC). Rats were exposed to 1-ppm AFB1 in feed for 14, 90, and 90 days plus a recovery 60-day, non-exposure period (150-day) timepoint. Isolated liver DNA was exome sequenced. We identified 172 sequence variants across all timepoints, of which 101 were non-synonymous variants. Well-annotated genes carried a diverse set of 29 non-synonymous mutations at 14 days, increasing to 39 mutations at 90 days and then decreasing to 33 mutations following the 60-day recovery. Gene Set Enrichment Analysis conducted on previously reported, available RNA expression data of the same exome sequenced archival samples identified altered transcripts in pathways associated with malignant transformation. These included HALLMARK gene sets associated with cell proliferation (MYC Targets Version 1 and Version 2, E2F targets), cell cycle (G2M checkpoint, mitotic spindle), cell death (apoptosis), and DNA damage (DNA repair, UV response Up, Reactive oxygen species) pathways. DriverNet Impact analysis integrated exome-seq and expression data to reveal somatic mutations in Mcm8, Bdp1, and Cct6a that may drive cancer formation. Connectivity with transcript expression changes identified these genes as the top-ranked candidate driver genes associated with hepatocellular transformation. In conclusion, exome sequencing revealed early somatic mutations that may play a role in cancer cell transformation that are translatable to aflatoxin-induced HCC.

Insights into Repeated Renal Injury Using RNA-Seq with Two New RPTEC Cell Lines.

Renal proximal tubule epithelial cells (RPTECs) are a primary site for kidney injury. We created two RPTEC lines from CD-1 mice immortalized with hTERT (human telomerase reverse transcriptase) or SV40 LgT antigen (Simian Virus 40 Large T antigen). Our hypothesis was that low-level, repeated exposure to subcytotoxic levels of 0.25-2.5 µM cisplatin (CisPt) or 12.5-100 µM aflatoxin B1 (AFB1) would activate distinctive genes and pathways in these two differently immortalized cell lines. RNA-seq showed only LgT cells responded to AFB1 with 1139 differentially expressed genes (DEGs) at 72 h. The data suggested that AFB1 had direct nephrotoxic properties on the LgT cells. However, both the cell lines responded to 2.5 µM CisPt from 3 to 96 h expressing 2000-5000 total DEGs. For CisPt, the findings indicated a coordinated transcriptional program of injury signals and repair from the expression of immune receptors with cytokine and chemokine secretion for leukocyte recruitment; robust expression of synaptic and substrate adhesion molecules (SAMs) facilitating the expression of neural and hormonal receptors, ion channels/transporters, and trophic factors; and the expression of nephrogenesis transcription factors. Pathway analysis supported the concept of a renal repair transcriptome. In summary, these cell lines provide in vitro models for the improved understanding of repeated renal injury and repair mechanisms. High-throughput screening against toxicant libraries should provide a wider perspective of their capabilities in nephrotoxicity.

Methionine metabolism is essential for SIRT1-regulated mouse embryonic stem cell maintenance and embryonic development.

Methionine metabolism is critical for epigenetic maintenance, redox homeostasis, and animal development. However, the regulation of methionine metabolism remains unclear. Here, we provide evidence that SIRT1, the most conserved mammalian NAD+-dependent protein deacetylase, is critically involved in modulating methionine metabolism, thereby impacting maintenance of mouse embryonic stem cells (mESCs) and subsequent embryogenesis. We demonstrate that SIRT1-deficient mESCs are hypersensitive to methionine restriction/depletion-induced differentiation and apoptosis, primarily due to a reduced conversion of methionine to S-adenosylmethionine. This reduction markedly decreases methylation levels of histones, resulting in dramatic alterations in gene expression profiles. Mechanistically, we discover that the enzyme converting methionine to S-adenosylmethionine in mESCs, methionine adenosyltransferase 2a (MAT2a), is under control of Myc and SIRT1. Consistently, SIRT1 KO embryos display reduced Mat2a expression and histone methylation and are sensitive to maternal methionine restriction-induced lethality, whereas maternal methionine supplementation increases the survival of SIRT1 KO newborn mice. Our findings uncover a novel regulatory mechanism for methionine metabolism and highlight the importance of methionine metabolism in SIRT1-mediated mESC maintenance and embryonic development.

Glucocorticoid receptor isoform-specific regulation of development, circadian rhythm, and inflammation in mice.

Glucocorticoids are primary stress hormones, and their synthetic derivatives are widely used clinically. The therapeutic efficacy of these steroids is limited by side effects and glucocorticoid resistance. Multiple glucocorticoid receptor (GR) isoforms are produced from a single gene by alternative translation initiation; however, the role individual isoforms play in tissue-specific responses to glucocorticoids is unknown. We have generated knockin mice that exclusively express the most active receptor isoform, GR-C3. GR-C3 knockin mice die at birth due to respiratory distress. Microarray analysis of fibroblasts from wild-type and GR-C3 mice indicated that most genes regulated by GR-C3 were unique to this isoform. Antenatal glucocorticoid administration rescued GR-C3 knockin mice from neonatal death. Dual-energy X-ray absorptiometry revealed no major alterations in body composition for rescued knockin mice. Rescued female, but not male, GR-C3 mice exhibited increased wheel running activity in the light portion of the day. LPS administration induced premature mortality in rescued GR-C3 knockin mice, and gene expression studies revealed a deficiency in the ability of GR-C3 to repress a large cohort of immune and inflammatory response genes. These findings demonstrate that specific GR translational isoforms can influence development, circadian rhythm, and inflammation through the regulation of distinct gene networks.-Oakley, R. H., Ramamoorthy, S., Foley, J. F., Busada, J. T., Lu, N. Z., Cidlowski, J. A. Glucocorticoid receptor isoform-specific regulation of development, circadian rhythm, and inflammation in mice.

Whole exome sequencing in the rat.

BACKGROUND: The rat genome was sequenced in 2004 with the aim to improve human health altered by disease and environmental influences through gene discovery and animal model validation. Here, we report development and testing of a probe set for whole exome sequencing (WES) to detect sequence variants in exons and UTRs of the rat genome. Using an in-silico approach, we designed probes targeting the rat exome and compared captured mutations in cancer-related genes from four chemically induced rat tumor cell lines (C6, FAT7, DSL-6A/C1, NBTII) to validated cancer genes in the human database, Catalogue of Somatic Mutations in Cancer (COSMIC) as well as normal rat DNA. Paired, fresh frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) liver tissue from naive rats were sequenced to confirm known dbSNP variants and identify any additional variants. RESULTS: Informatics analysis of available gene annotation from rat RGSC6.0/rn6 RefSeq and Ensembl transcripts provided 223,636 unique exons representing a total of 26,365 unique genes and untranslated regions. Using this annotation and the Rn6 reference genome, an in-silico probe design generated 826,878 probe sequences of which 94.2% were uniquely aligned to the rat genome without mismatches. Further informatics analysis revealed 25,249 genes (95.8%) covered by at least one probe and 23,603 genes (93.5%) had every exon covered by one or more probes. We report high performance metrics from exome sequencing of our probe set and Sanger validation of annotated, highly relevant, cancer gene mutations as cataloged in the human COSMIC database, in addition to several exonic variants in cancer-related genes. CONCLUSIONS: An in-silico probe set was designed to enrich the rat exome from isolated DNA. The platform was tested on rat tumor cell lines and normal FF and FFPE liver tissue. The method effectively captured target exome regions in the test DNA samples with exceptional sensitivity and specificity to obtain reliable sequencing data representing variants that are likely chemically induced somatic mutations. Genomic discovery conducted by means of high throughput WES queries should benefit investigators in discovering rat genomic variants in disease etiology and in furthering human translational research.

Histology Atlas of the Developing Prenatal and Postnatal Mouse Central Nervous System, with Emphasis on Prenatal Days E7.5 to E18.5.

Evaluation of the central nervous system (CNS) in the developing mouse presents unique challenges, given the complexity of ontogenesis, marked structural reorganization over very short distances in 3 dimensions each hour, and numerous developmental events susceptible to genetic and environmental influences. Developmental defects affecting the brain and spinal cord arise frequently both in utero and perinatally as spontaneous events, following teratogen exposure, and as sequelae to induced mutations and thus are a common factor in embryonic and perinatal lethality in many mouse models. Knowledge of normal organ and cellular architecture and differentiation throughout the mouse's life span is crucial to identify and characterize neurodevelopmental lesions. By providing a well-illustrated overview summarizing major events of normal in utero and perinatal mouse CNS development with examples of common developmental abnormalities, this annotated, color atlas can be used to identify normal structure and histology when phenotyping genetically engineered mice and will enhance efforts to describe and interpret brain and spinal cord malformations as causes of mouse embryonic and perinatal lethal phenotypes. The schematics and images in this atlas illustrate major developmental events during gestation from embryonic day (E)7.5 to E18.5 and after birth from postnatal day (P)1 to P21.

Preparation of High-quality Hematoxylin and Eosin-stained Sections from Rodent Mammary Gland Whole Mounts for Histopathologic Review.

Identifying environmental exposures that cause adverse mammary gland outcomes in rodents is a first step in disease prevention in humans and domestic pets. "Whole mounts" are an easy and inexpensive tissue preparation method that can elucidate typical or abnormal mammary gland morphology in rodent studies. Here, we propose procedures to facilitate the use of whole mounts for histological identification of grossly noted tissue alterations. We noted lesions in mammary whole mounts from 14-month-old CD-1 mice that were not found in the contralateral gland hematoxylin and eosin (H&E)-stained section. Whole mounts were removed from the slide and carefully processed to produce high-quality histological sections that mirrored the quality of the original H&E-stained section in order to properly diagnose the unidentified gross abnormalities. Incorporation of this method into testing protocols that focus on human relevant chemical and endocrine disruptors exposure will increase the chances of identifying lesions in the gland and reduce the risk of false negative findings. This method can be especially invaluable when lesions are not always palpable during the course of the study or visible at necropsy, or when a single cross section of the mammary gland is otherwise used for detecting lesions.

Histology Atlas of the Developing Mouse Hepatobiliary Hemolymphatic Vascular System with Emphasis on Embryonic Days 11.5-18.5 and Early Postnatal Development.

A critical event in embryo development is the proper formation of the vascular system, of which the hepatobiliary system plays a pivotal role. This has led researchers to use transgenic mice to identify the critical steps involved in developmental disorders associated with the hepatobiliary vascular system. Vascular development is dependent upon normal vasculogenesis, angiogenesis, and the transformation of vessels into their adult counterparts. Any alteration in vascular development has the potential to cause deformities or embryonic death. Numerous publications describe specific stages of vascular development relating to various organs, but a single resource detailing the stage-by-stage development of the vasculature pertaining to the hepatobiliary system has not been available. This comprehensive histology atlas provides hematoxylin & eosin and immunohistochemical-stained sections of the developing mouse blood and lymphatic vasculature with emphasis on the hepatobiliary system between embryonic days (E) 11.5-18.5 and the early postnatal period. Additionally, this atlas includes a 3-dimensional video representation of the E18.5 mouse venous vasculature. One of the most noteworthy findings of this atlas is the identification of the portal sinus within the mouse, which has been erroneously misinterpreted as the ductus venosus in previous publications. Although the primary purpose of this atlas is to identify normal hepatobiliary vascular development, potential embryonic abnormalities are also described.

Molecular Changes in the Nasal Cavity after N, N-dimethyl-p-toluidine Exposure.

N, N-dimethyl-p-toluidine (DMPT; Cas No. 99-97-8), an accelerant for methyl methacrylate monomers in medical devices, is a nasal cavity carcinogen according to a 2-yr cancer study of male and female F344/N rats, with the nasal tumors arising from the transitional cell epithelium. In this study, we exposed male F344/N rats for 5 days to DMPT (0, 1, 6, 20, 60, or 120 mg/kg [oral gavage]) to explore the early changes in the nasal cavity after short-term exposure. Lesions occurred in the nasal cavity including hyperplasia of transitional cell epithelium (60 and 120 mg/kg). Nasal tissue was rapidly removed and preserved for subsequent laser capture microdissection and isolation of the transitional cell epithelium (0 and 120 mg/kg) for transcriptomic studies. DMPT transitional cell epithelium gene transcript patterns were characteristic of an antioxidative damage response (e.g., Akr7a3, Maff, and Mgst3), cell proliferation, and decrease in signals for apoptosis. The transcripts of amino acid transporters were upregulated (e.g., Slc7a11). The DMPT nasal transcript expression pattern was similar to that found in the rat nasal cavity after formaldehyde exposure, with over 1,000 transcripts in common. Molecular changes in the nasal cavity after DMPT exposure suggest that oxidative damage is a mechanism of the DMPT toxic and/or carcinogenic effects.

Mammary Gland Evaluation in Juvenile Toxicity Studies: Temporal Developmental Patterns in the Male and Female Harlan Sprague-Dawley Rat.

There are currently no reports describing mammary gland development in the Harlan Sprague-Dawley (HSD) rat, the current strain of choice for National Toxicology Program (NTP) testing. Our goals were to empower the NTP, contract labs, and other researchers in understanding and interpreting chemical effects in this rat strain. To delineate similarities/differences between the female and male mammary gland, data were compiled starting on embryonic day 15.5 through postnatal day 70. Mammary gland whole mounts, histology sections, and immunohistochemically stained tissues for estrogen, progesterone, and androgen receptors were evaluated in both sexes; qualitative and quantitative differences are highlighted using a comprehensive visual timeline. Research on endocrine disrupting chemicals in animal models has highlighted chemically induced mammary gland anomalies that may potentially impact human health. In order to investigate these effects within the HSD strain, 2,3,7,8-tetrachlorodibenzo-p-dioxin, diethylstilbestrol, or vehicle control was gavage dosed on gestation day 15 and 18 to demonstrate delayed, accelerated, and control mammary gland growth in offspring, respectively. We provide illustrations of normal and chemically altered mammary gland development in HSD male and female rats to help inform researchers unfamiliar with the tissue and may facilitate enhanced evaluation of both male and female mammary glands in juvenile toxicity studies.

Polg2 is essential for mammalian embryogenesis and is required for mtDNA maintenance.

Mammalian mitochondrial DNA (mtDNA) is replicated by the heterotrimeric Pol γ comprised of a single catalytic subunit, encoded by Polg, and a homodimeric accessory subunit encoded by the Polg2 gene. While the catalytic subunit has been shown to be essential for embryo development, genetic data regarding the accessory subunit are lacking in mammalian systems. Here, we describe the generation of heterozygous (Polg2(+/-)) and homozygous (Polg2(-/-)) knockout (KO) mice. Polg2(+/-) mice are haplosufficient and develop normally with no discernable difference in mitochondrial function through 2 years of age. In contrast, the Polg2(-/-) is embryonic lethal at day 8.0-8.5 p.c. with concomitant loss of mtDNA and mtDNA gene products. Electron microscopy shows severe ultra-structural defects and loss of organized cristae in mitochondria of the Polg2(-/-) embryos as well as an increase in lipid accumulation compared with both wild-type (WT) and Polg2(+/-) embryos. Our data indicate that Polg2 function is critical to mammalian embryogenesis and mtDNA replication, and that a single copy of Polg2 is sufficient to sustain life.

Genomic Profiling Reveals Unique Molecular Alterations in Hepatoblastomas and Adjacent Hepatocellular Carcinomas in B6C3F1 Mice.

The cell of origin of hepatoblastoma (HB) in humans and mice is unknown; it is hypothesized to be a transformed hepatocyte, oval cell, or hepatic progenitor cell. In mice, current dogma is that HBs arise from preexisting hepatocellular neoplasms as a result of further neoplastic transformation. However, there is little evidence supporting this direct relationship. To better understand the relationship between hepatocellular carcinoma (HCC) and HB and determine molecular similarities between mouse and human HB, global gene expression analysis and targeted mutation analysis were performed using HB, HCC, and adjacent liver from the same animals in a recent National Toxicology Program bioassay. There were significant differences in Hras and Ctnnb1 mutation spectra, and by microarray, HBs showed dysregulation of embryonic development, stem cell pluripotency, and genomic imprinting compared to HCC. Meta-analysis showed similarities between HB, early mouse embryonic liver, and hepatocyte-derived stem/progenitor cells compared to HCC. Our data show that there are striking differences between HB and HCC and suggest that HB is a significantly different entity that may arise from a hepatic precursor cell. Furthermore, mouse HB is similar to the human disease at the pathway level and therefore is likely a relevant model for evaluating human cancer hazard.

ATP binding cassette transporter G1 deletion induces IL-17-dependent dysregulation of pulmonary adaptive immunity.

Mice with genetic deletion of the cholesterol transporter ATP binding cassette G1 (ABCG1) have pulmonary lipidosis and enhanced innate immune responses in the airway. Whether ABCG1 regulates adaptive immune responses to the environment is unknown. To this end, Abcg1(+/+) and Abcg1(-/-) mice were sensitized to OVA via the airway using low-dose LPS as an adjuvant, and then challenged with OVA aerosol. Naive Abcg1(-/-) mice displayed increased B cells, CD4(+) T cells, CD8(+) T cells, and dendritic cells (DCs) in lung and lung-draining mediastinal lymph nodes, with lung CD11b(+) DCs displaying increased CD80 and CD86. Upon allergen sensitization and challenge, the Abcg1(-/-) airway, compared with Abcg1(+/+), displayed reduced Th2 responses (IL-4, IL-5, eosinophils), increased neutrophils and IL-17, but equivalent airway hyperresponsiveness. Reduced Th2 responses were also found using standard i.p. OVA sensitization with aluminum hydroxide adjuvant. Mediastinal lymph nodes from airway-sensitized Abcg1(-/-) mice produced reduced IL-5 upon ex vivo OVA challenge. Abcg1(-/-) CD4(+) T cells displayed normal ex vivo differentiation, whereas Abcg1(-/-) DCs were found paradoxically to promote Th2 polarization. Th17 cells, IL-17(+) γδT cells, and IL-17(+) neutrophils were all increased in Abcg1(-/-) lungs, suggesting Th17 and non-Th17 sources of IL-17 excess. Neutralization of IL-17 prior to challenge normalized eosinophils and reduced neutrophilia in the Abcg1(-/-) airway. We conclude that Abcg1(-/-) mice display IL-17-mediated suppression of eosinophilia and enhancement of neutrophilia in the airway following allergen sensitization and challenge. These findings identify ABCG1 as a novel integrator of cholesterol homeostasis and adaptive immune programs.

Testing an aflatoxin B1 gene signature in rat archival tissues.

Archival tissues from laboratory studies represent a unique opportunity to explore the relationship between genomic changes and agent-induced disease. In this study, we evaluated the applicability of qPCR for detecting genomic changes in formalin-fixed, paraffin-embedded (FFPE) tissues by determining if a subset of 14 genes from a 90-gene signature derived from microarray data and associated with eventual tumor development could be detected in archival liver, kidney, and lung of rats exposed to aflatoxin B1 (AFB1) for 90 days in feed at 1 ppm. These tissues originated from the same rats used in the microarray study. The 14 genes evaluated were Adam8, Cdh13, Ddit4l, Mybl2, Akr7a3, Akr7a2, Fhit, Wwox, Abcb1b, Abcc3, Cxcl1, Gsta5, Grin2c, and the C8orf46 homologue. The qPCR FFPE liver results were compared to the original liver microarray data and to qPCR results using RNA from fresh frozen liver. Archival liver paraffin blocks yielded 30 to 50 µg of degraded RNA that ranged in size from 0.1 to 4 kB. qPCR results from FFPE and fresh frozen liver samples were positively correlated (p ≤ 0.05) by regression analysis and showed good agreement in direction and proportion of change with microarray data for 11 of 14 genes. All 14 transcripts could be amplified from FFPE kidney RNA except the glutamate receptor gene Grin2c; however, only Abcb1b was significantly upregulated from control. Abundant constitutive transcripts, S18 and ß-actin, could be amplified from lung FFPE samples, but the narrow RNA size range (25-500 bp length) prevented consistent detection of target transcripts. Overall, a discrete gene signature derived from prior transcript profiling and representing cell cycle progression, DNA damage response, and xenosensor and detoxication pathways was successfully applied to archival liver and kidney by qPCR and indicated that gene expression changes in response to subchronic AFB1 exposure occurred predominantly in the liver, the primary target for AFB1-induced tumors. We conclude that an evaluation of gene signatures in archival tissues can be an important toxicological tool for evaluating critical molecular events associated with chemical exposures.

Endothelial expression of human cytochrome P450 epoxygenase CYP2C8 increases susceptibility to ischemia-reperfusion injury in isolated mouse heart.

Cytochrome P450 (CYP) epoxygenases CYP2C8 and CYP2J2 generate epoxyeicosatrienoic acids (EETs) from arachidonic acid. Mice with expression of CYP2J2 in cardiomyocytes (αMHC-CYP2J2 Tr) or treated with synthetic EETs have increased functional recovery after ischemia/reperfusion (I/R); however, no studies have examined the role of cardiomyocyte- vs. endothelial-derived EETs or compared the effects of different CYP epoxygenase isoforms in the ischemic heart. We generated transgenic mice with increased endothelial EET biosynthesis (Tie2-CYP2C8 Tr and Tie2-CYP2J2 Tr) or EET hydrolysis (Tie2-sEH Tr). Compared to wild-type (WT), αMHC-CYP2J2 Tr hearts showed increased recovery of left ventricular developed pressure (LVDP) and decreased infarct size after I/R. In contrast, LVDP recovery and infarct size were unchanged in Tie2-CYP2J2 Tr and Tie2-sEH Tr hearts. Surprisingly, compared to WT, Tie2-CYP2C8 Tr hearts had significantly reduced LVDP recovery (from 21 to 14%) and increased infarct size after I/R (from 51 to 61%). Tie2-CYP2C8 Tr hearts also exhibited increased reactive oxygen species (ROS) generation, dihydroxyoctadecenoic acid (DiHOME) formation, and coronary resistance after I/R. ROS scavengers and CYP2C8 inhibition reversed the detrimental effects of CYP2C8 expression in Tie2-CYP2C8 Tr hearts. Treatment of WT hearts with 250 nM 9,10-DiHOME decreased LVDP recovery compared to vehicle (16 vs. 31%, respectively) and increased coronary resistance after I/R. These data demonstrate that increased ROS generation and enhanced DiHOME synthesis by endothelial CYP2C8 impair functional recovery and mask the beneficial effects of increased EET production following I/R.

Endothelial CYP epoxygenase overexpression and soluble epoxide hydrolase disruption attenuate acute vascular inflammatory responses in mice.

Cytochrome P-450 (CYP)-derived epoxyeicosatrienoic acids (EETs) possess potent anti-inflammatory effects in vitro. However, the effect of increased CYP-mediated EET biosynthesis and decreased soluble epoxide hydrolase (sEH, Ephx2)-mediated EET hydrolysis on vascular inflammation in vivo has not been rigorously investigated. Consequently, we characterized acute vascular inflammatory responses to endotoxin in transgenic mice with endothelial expression of the human CYP2J2 and CYP2C8 epoxygenases and mice with targeted disruption of Ephx2. Compared to wild-type controls, CYP2J2 transgenic, CYP2C8 transgenic, and Ephx2(-/-) mice each exhibited a significant attenuation of endotoxin-induced activation of nuclear factor (NF)-κB signaling, cellular adhesion molecule, chemokine and cytokine expression, and neutrophil infiltration in lung in vivo. Furthermore, attenuation of endotoxin-induced NF-κB activation and cellular adhesion molecule and chemokine expression was observed in primary pulmonary endothelial cells isolated from CYP2J2 and CYP2C8 transgenic mice. This attenuation was inhibited by a putative EET receptor antagonist and CYP epoxygenase inhibitor, directly implicating CYP epoxygenase-derived EETs with the observed anti-inflammatory phenotype. Collectively, these data demonstrate that potentiation of the CYP epoxygenase pathway by either increased endothelial EET biosynthesis or globally decreased EET hydrolysis attenuates NF-κB-dependent vascular inflammatory responses in vivo and may serve as a viable anti-inflammatory therapeutic strategy.

A Novel Mouse Model to Analyze Non-Genomic ERα Physiological Actions.

Nongenomic effects of estrogen receptor α (ERα) signaling have been described for decades. Several distinct animal models have been generated previously to analyze the nongenomic ERα signaling (eg, membrane-only ER, and ERαC451A). However, the mechanisms and physiological processes resulting solely from nongenomic signaling are still poorly understood. Herein, we describe a novel mouse model for analyzing nongenomic ERα actions named H2NES knock-in (KI). H2NES ERα possesses a nuclear export signal (NES) in the hinge region of ERα protein resulting in exclusive cytoplasmic localization that involves only the nongenomic action but not nuclear genomic actions. We generated H2NESKI mice by homologous recombination method and have characterized the phenotypes. H2NESKI homozygote mice possess almost identical phenotypes with ERα null mice except for the vascular activity on reendothelialization. We conclude that ERα-mediated nongenomic estrogenic signaling alone is insufficient to control most estrogen-mediated endocrine physiological responses; however, there could be some physiological responses that are nongenomic action dominant. H2NESKI mice have been deposited in the repository at Jax (stock no. 032176). These mice should be useful for analyzing nongenomic estrogenic responses and could expand analysis along with other ERα mutant mice lacking membrane-bound ERα. We expect the H2NESKI mouse model to aid our understanding of ERα-mediated nongenomic physiological responses and serve as an in vivo model for evaluating the nongenomic action of various estrogenic agents.

Ventilation defects observed with hyperpolarized 3He magnetic resonance imaging in a mouse model of acute lung injury.

Regions of diminished ventilation are often evident during functional pulmonary imaging studies, including hyperpolarized gas magnetic resonance imaging (MRI), positron emission tomography, and computed tomography (CT). The objective of this study was to characterize the hypointense regions observed via (3)He MRI in a murine model of acute lung injury. LPS at doses ranging from 15-50 µg was intratracheally administered to C57BL/6 mice under anesthesia. Four hours after exposure to either LPS or saline vehicle, mice were imaged via hyperpolarized (3)He MRI. All images were evaluated to identify regions of hypointense signals. Lungs were then characterized by conventional histology, or used to obtain tissue samples from regions of normal and hypointense (3)He signals and analyzed for cytokine content. The characterization of (3)He MRI images identified three distinct types of hypointense patterns: persistent defects, atelectatic defects, and dorsal lucencies. Persistent defects were associated with the administration of LPS. The number of persistent defects depended on the dose of LPS, with a significant increase in mean number of defects in 30-50-µg LPS-dosed mice versus saline-treated control mice. Atelectatic defects predominated in LPS-dosed mice under conditions of low-volume ventilation, and could be reversed with deep inspiration. Dorsal lucencies were present in nearly all mice studied, regardless of the experimental conditions, including control animals that did not receive LPS. A comparison of (3)He MRI with histopathology did not identify tissue abnormalities in regions of low (3)He signal, with the exception of a single region of atelectasis in one mouse. Furthermore, no statistically significant differences were evident in concentrations of IL-1ß, IL-6, macrophage inflammatory protein (MIP)-1α, MIP-2, chemokine (C-X-C motif) ligand 1 (KC), TNFα, and monocyte chemotactic protein (MCP)-1 between hypointense and normally ventilated lung regions in LPS-dosed mice. Thus, this study defines the anatomic, functional, and biochemical characteristics of ventilation defects associated with the administration of LPS in a murine model of acute lung injury.

Embryonic expression of cyclooxygenase-2 causes malformations in axial skeleton.

Cyclooxygenases (COXs) have important functions in various physiological and pathological processes. COX-2 expression is highly induced by a variety of stimuli and is observed during certain periods of embryonic development. In this report, the direct effect of COX-2 expression on embryonic development is examined in a novel COX-2 transgenic mouse model that ubiquitously expresses human COX-2 from the early stages of embryonic development. COX-2 transgenic fetuses exhibit severe skeletal malformations and die shortly after birth. Skeletal malformations are localized along the entire vertebral column and rib cage and are linked to defective formation of cartilage anlagen. The cartilage anlagen of axial skeleton fail to properly develop in transgenic embryos because of impaired precartilaginous sclerotomal condensation, which results from the reduction of cell number in the sclerotome. Despite the ubiquitous expression of COX-2, the number of apoptotic cells is highly increased in the sclerotome of transgenic embryos but not in other tissues, suggesting that it is a tissue-specific response. Therefore, the loss of sclerotomal cells due to an increased apoptosis is probably responsible for axial skeletal malformations in transgenic fetuses. In addition, the sclerotomal accumulation of p53 protein is observed in transgenic embryos, suggesting that COX-2 may induce apoptosis via the up-regulation of p53. Our results demonstrate that the aberrant COX-2 signaling during embryonic development is teratogenic and suggest a possible association of COX-2 with fetal malformations of unknown etiology.

Endothelial expression of human cytochrome P450 epoxygenases lowers blood pressure and attenuates hypertension-induced renal injury in mice.

Renal cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) regulate sodium transport and blood pressure. Although endothelial CYP-derived EETs are potent vasodilators, their contribution to the regulation of blood pressure remains unclear. Consequently, we developed transgenic mice with endothelial expression of the human CYP2J2 and CYP2C8 epoxygenases to increase endothelial EET biosynthesis. Compared to wild-type littermate controls, an attenuated afferent arteriole constrictor response to endothelin-1 and enhanced dilator response to acetylcholine was observed in CYP2J2 and CYP2C8 transgenic mice. CYP2J2 and CYP2C8 transgenic mice demonstrated modestly, but not significantly, lower mean arterial pressure under basal conditions compared to wild-type controls. However, mean arterial pressure was significantly lower in both CYP2J2 and CYP2C8 transgenic mice during coadministration of N-nitro-l-arginine methyl ester and indomethacin. In a separate experiment, a high-salt diet and subcutaneous angiotensin II was administered over 4 wk. The angiotensin/high-salt-induced increase in systolic blood pressure, proteinuria, and glomerular injury was significantly attenuated in CYP2J2 and CYP2C8 transgenic mice compared to wild-type controls. Collectively, these data demonstrate that increased endothelial CYP epoxygenase expression attenuates afferent arteriolar constrictor reactivity and hypertension-induced increases in blood pressure and renal injury in mice. We conclude that endothelial CYP epoxygenase function contributes to the regulation of blood pressure.