A review of the toxicology of oil in vertebrates: what we have learned following the Deepwater Horizon oil spill.

In the wake of the Deepwater Horizon (DWH) oil spill, a number of government agencies, academic institutions, consultants, and nonprofit organizations conducted lab- and field-based research to understand the toxic effects of the oil. Lab testing was performed with a variety of fish, birds, turtles, and vertebrate cell lines (as well as invertebrates); field biologists conducted observations on fish, birds, turtles, and marine mammals; and epidemiologists carried out observational studies in humans. Eight years after the spill, scientists and resource managers held a workshop to summarize the similarities and differences in the effects of DWH oil on vertebrate taxa and to identify remaining gaps in our understanding of oil toxicity in wildlife and humans, building upon the cross-taxonomic synthesis initiated during the Natural Resource Damage Assessment. Across the studies, consistency was found in the types of toxic response observed in the different organisms. Impairment of stress responses and adrenal gland function, cardiotoxicity, immune system dysfunction, disruption of blood cells and their function, effects on locomotion, and oxidative damage were observed across taxa. This consistency suggests conservation in the mechanisms of action and disease pathogenesis. From a toxicological perspective, a logical progression of impacts was noted: from molecular and cellular effects that manifest as organ dysfunction, to systemic effects that compromise fitness, growth, reproductive potential, and survival. From a clinical perspective, adverse health effects from DWH oil spill exposure formed a suite of signs/symptomatic responses that at the highest doses/concentrations resulted in multi-organ system failure.

Epigenetic Regulation by Agonist-Specific Aryl Hydrocarbon Receptor Recruitment of Metastasis-Associated Protein 2 Selectively Induces Stanniocalcin 2 Expression.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates a plethora of target genes. Historically, the AhR has been studied as a regulator of xenobiotic metabolizing enzyme genes, notably cytochrome P4501A1 encoded by CYP1A1, in response to the exogenous prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AhR activity depends on its binding to the xenobiotic response element (XRE) in partnership with the AhR nuclear translocator (Arnt). Recent studies identified stanniocalcin 2 (Stc2) as a novel AhR target gene responsive to the endogenous AhR agonist cinnabarinic acid (CA). CA-dependent AhR-XRE-mediated Stc2 upregulation is responsible for cytoprotection against ectoplasmic reticulum/oxidative stress-induced apoptosis both in vitro and in vivo. Significantly, CA but not TCDD induces expression of Stc2 in hepatocytes. In contrast to TCDD, CA is unable to induce the CYP1A1 gene, thus revealing an AhR agonist-specific mutually exclusive dichotomous transcriptional response. Studies reported here provide a mechanistic explanation for this differential response by identifying an interaction between the AhR and the metastasis-associated protein 2 (MTA2). Moreover, the AhR-MTA2 interaction is CA-dependent and results in MTA2 recruitment to the Stc2 promoter, concomitant with agonist-specific epigenetic modifications targeting histone H4 lysine acetylation. The results demonstrate that histone H4 acetylation is absolutely dependent on CA-induced AhR and MTA2 recruitment to the Stc2 regulatory region and induced Stc2 gene expression, which in turn confers cytoprotection to liver cells exposed to chemical insults.

The MET Receptor Tyrosine Kinase Confers Repair of Murine Pancreatic Acinar Cells following Acute and Chronic Injury.

Acinar cells represent the primary target in necroinflammatory diseases of the pancreas, including pancreatitis. The signaling pathways guiding acinar cell repair and regeneration following injury remain poorly understood. The purpose of this study was to determine the importance of Hepatocyte Growth Factor Receptor/MET signaling as an intrinsic repair mechanism for acinar cells following acute damage and chronic alcohol-associated injury. Here, we generated mice with targeted deletion of MET in adult acinar cells (MET-/-). Acute and repetitive pancreatic injury was induced in MET-/- and control mice with cerulein, and chronic injury by feeding mice Lieber-DeCarli diets containing alcohol with or without enhancement of repetitive pancreatic injury. We examined the exocrine pancreas of these mice histologically for acinar death, edema, inflammation and collagen deposition and changes in the transcriptional program. We show that MET expression is relatively low in normal adult pancreas. However, MET levels were elevated in ductal and acinar cells in human pancreatitis specimens, consistent with a role for MET in an adaptive repair mechanism. We report that genetic deletion of MET in adult murine acinar cells was linked to increased acinar cell death, chronic inflammation and delayed recovery (regeneration) of pancreatic exocrine tissue. Notably, increased pancreatic collagen deposition was detected in MET knockout mice following repetitive injury as well alcohol-associated injury. Finally, we identified specific alterations of the pancreatic transcriptome associated with MET signaling during injury, involved in tissue repair, inflammation and endoplasmic reticulum stress. Together, these data demonstrate the importance of MET signaling for acinar repair and regeneration, a novel finding that could attenuate the symptomology of pancreatic injury.

Targeted proteomics for biomarker discovery and validation of hepatocellular carcinoma in hepatitis C infected patients.

Hepatocellular carcinoma (HCC)-related mortality is high because early detection modalities are hampered by inaccuracy, expense and inherent procedural risks. Thus there is an urgent need for minimally invasive, highly specific and sensitive biomarkers that enable early disease detection when therapeutic intervention remains practical. Successful therapeutic intervention is predicated on the ability to detect the cancer early. Similar unmet medical needs abound in most fields of medicine and require novel methodological approaches. Proteomic profiling of body fluids presents a sensitive diagnostic tool for early cancer detection. Here we describe such a strategy of comparative proteomics to identify potential serum-based biomarkers to distinguish high-risk chronic hepatitis C virus infected patients from HCC patients. In order to compensate for the extraordinary dynamic range in serum proteins, enrichment methods that compress the dynamic range without surrendering proteome complexity can help minimize the problems associated with many depletion methods. The enriched serum can be resolved using 2D-difference in-gel electrophoresis and the spots showing statistically significant changes selected for identification by liquid chromatography-tandem mass spectrometry. Subsequent quantitative verification and validation of these candidate biomarkers represent an obligatory and rate-limiting process that is greatly enabled by selected reaction monitoring (SRM). SRM is a tandem mass spectrometry method suitable for identification and quantitation of target peptides within complex mixtures independent on peptide-specific antibodies. Ultimately, multiplexed SRM and dynamic multiple reaction monitoring can be utilized for the simultaneous analysis of a biomarker panel derived from support vector machine learning approaches, which allows monitoring a specific disease state such as early HCC. Overall, this approach yields high probability biomarkers for clinical validation in large patient cohorts and represents a strategy extensible to many diseases.

Ah receptor-mediated suppression of liver regeneration through NC-XRE-driven p21Cip1 expression.

Previous studies in hepatocyte-derived cell lines and the whole liver established that the aryl hydrocarbon receptor (AhR) can disrupt G1-phase cell cycle progression following exposure to persistent AhR agonists, such as TCDD (dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin). Growth arrest was attributed to inhibition of G1-phase cyclin-dependent kinase 2 (CDK2) activity. The present study examined the effect of TCDD exposure on liver regeneration following 70% partial hepatectomy in mice lacking the Cip/Kip inhibitors p21(Cip1) or p27(Kip1) responsible for regulating CDK2 activity. Assessment of the regenerative process in wild-type, p21(Cip1) knockout, and p27(Kip1) knockout mice confirmed that TCDD-induced inhibition of liver regeneration is entirely dependent on p21(Cip1) expression. Compared with wild-type mice, the absence of p21(Cip1) expression completely abrogated the TCDD inhibition, and accelerated hepatocyte progression through G1 phase during the regenerative process. Analysis of the transcriptional response determined that increased p21(Cip1) expression during liver regeneration involved an AhR-dependent mechanism. Chromatin immunoprecipitation studies revealed that p21(Cip1) induction required AhR binding to the newly characterized nonconsensus xenobiotic response element, in conjunction with the tumor suppressor protein Kruppel-like factor 6 functioning as an AhR binding partner. The evidence also suggests that AhR functionality following partial hepatectomy is dependent on a p21(Cip1)-regulated signaling process, intimately linking AhR biology to the G1-phase cell cycle program.

The tumor suppressor Kruppel-like factor 6 is a novel aryl hydrocarbon receptor DNA binding partner.

The aryl hydrocarbon receptor (AhR) is a ligand-mediated basic helix-loop-helix transcription factor of the Per/Arnt/Sim family that regulates adaptive and toxic responses to a variety of chemical pollutants, including polycyclic aromatic hydrocarbons and halogenated aromatic hydrocarbons, most notably 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Ligand activation leads to AhR nuclear translocation and binding to a xenobiotic response element (XRE) in association with the Arnt to regulate gene expression. Several recent genome-wide transcriptional studies identified numerous AhR target genes that lack the canonical XRE recognition site in the promoter regions. Characterization of one such target gene, the plasminogen activator inhibitor 1, identified a novel nonconsensus XRE (NC-XRE) that confers TCDD responsiveness independently of the Arnt protein. Studies reported here show that the NC-XRE is a recognition site for the AhR and a new binding partner, the Kruppel-like factor (KLF) family member KLF6. In vivo chromatin immunoprecipitations and in vitro DNA binding studies demonstrate that the AhR and KLF6 proteins form an obligatory heterodimer necessary for NC-XRE binding. Mutational analyses show that the protein-protein interactions involve the AhR C terminus and KLF6 N terminus, respectively. Moreover, NC-XRE binding depends on the 5' basic region in KLF6 rather than the previously characterized zinc finger DNA binding domain. Collectively, the results unmask a novel AhR signaling mechanism distinct from the canonical XRE-driven process that will enrich our future understanding of AhR biology.

The activated aryl hydrocarbon receptor synergizes mitogen-induced murine liver hyperplasia.

Mechanisms of hepatocyte proliferation triggered by tissue loss are distinguishable from those that promote proliferation in the intact liver in response to mitogens. Previous studies demonstrate that exogenous activation of the aryl hydrocarbon receptor (AhR), a soluble ligand-activated transcription factor in the basic helix-loop-helix family of proteins, suppresses compensatory liver regeneration elicited by surgical partial hepatectomy. The goal of the present study was to determine how AhR activation modulates hepatocyte cell cycle progression in the intact liver following treatment with the hepatomitogen, 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP). Mice were pretreated with the exogenous AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 24h prior to treatment with TCPOBOP (3 mg/kg).). In contrast to the suppressive effects of AhR activation observed during compensatory regeneration, TCDD pretreatment resulted in a 30-50% increase in hepatocyte proliferation in the intact liver of TCPOBOP-treated mice. Although pretreatment with TCDD suppressed CDK2 kinase activity and increased the association of CDK2 with negative regulatory proteins p21Cip1 and p27Kip1, a corresponding increase in CDK4/cyclin D1 association and CDK4 activity which culminated in enhanced phosphorylation of retinoblastoma protein, consistent with the increased proliferative response. These findings are in stark contrast to previous observations that the activated AhR can suppress hepatocyte proliferation in vivo and reveal a new complexity to AhR-mediated cell cycle control.

Requirement of biphasic calcium release from the endoplasmic reticulum for Fas-mediated apoptosis.

Fas receptor is a member of the tumor necrosis factor-alpha family of death receptors that mediate physiologic apoptotic signaling. To investigate the molecular mechanisms regulating calcium mobilization during Fas-mediated apoptosis, we have analyzed the sequential steps leading to altered calcium homeostasis and cell death in response to activation of the Fas receptor. We show that Fas-mediated apoptosis requires endoplasmic reticulum-mediated calcium release in a mechanism dependent on phospholipase C-gamma1 (PLC-gamma1) activation and Ca2+ release from inositol 1,4,5-trisphosphate receptor (IP3R) channels. The kinetics of Ca2+ release were biphasic, demonstrating a rapid elevation caused by PLC-gamma1 activation and a delayed and sustained increase caused by cytochrome c binding to IP3R. Blocking either phase of Ca2+ mobilization was cytoprotective, highlighting PLC-gamma1 and IP3R as possible therapeutic targets for disorders associated with Fas signaling.

Sustained aryl hydrocarbon receptor activity attenuates liver regeneration.

In hepatocyte-derived cell lines, either loss of aryl hydrocarbon receptor (AhR) function or treatment with a persistent AhR agonist such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can disrupt G1 phase cell cycle progression. The present study used liver regeneration to explore mechanistically how AhR activity modulates hepatocyte proliferation in vivo. Treatment of mice with 20 mug/kg TCDD 1 day before 70% partial hepatectomy (PH) resulted in a 50 to 75% suppression in liver regeneration. Impaired proliferation was not associated with changes in levels of interleukin-6 or tumor necrosis factor-alpha, which prime quiescent hepatocytes to enter G1 phase. In fact, administration of TCDD 12 h after PH, a period well beyond the priming phase, still induced the G1 arrest. Decreased proliferation in TCDD-treated mice correlated with reduced cyclin-dependent kinase-2 (CDK2) activity, a pivotal regulator of G1/S phase transition. In contrast to observations made in cell culture, suppressed CDK2 activity was not strictly associated with increased binding of the CDK2 inhibitors p21Cip1 or p27Kip1. However, TCDD decreased levels of cyclin E binding to CDK2, despite normal cyclin E expression. The evidence also suggests that TCDD-induced hepatic growth arrest depends upon sustained AhR activity because transient AhR activation in response to endogenous queues failed to suppress the regenerative response. These findings establish a functional role for the AhR in regulating normal cell cycle control during liver regeneration.

Multiple mechanisms are involved in Ah receptor-mediated cell cycle arrest.

The liver is the only solid organ that can respond to major tissue loss or damage by regeneration to restore liver biomass. The aryl hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can disrupt the regenerative process, as evidenced by suppression of DNA synthesis in rat primary hepatocytes in culture and in vivo liver regeneration after partial hepatectomy. Independent observations demonstrated that AhR-mediated G(1) phase cell cycle arrest depends on an interaction with the retinoblastoma tumor suppressor protein (pRb), but differences exist regarding proposed mechanisms of action. Two distinct models have been proposed, one supporting the AhR-pRb interaction functioning in corepression of E2F activity and the other favoring an AhR-pRb interaction participating in transcriptional coactivation of genes encoding G(1) phase regulatory proteins. In the present study, experiments in rat hepatoma cells using dominant-negative DNA-binding-defective AhR and Ah receptor nuclear translocator (Arnt) mutants provided evidence that TCDD-induced AhR-mediated G(1) arrest is only partially regulated by direct AhR transcriptional activity, suggesting that both coactivation and corepression are involved. Studies using a small interfering RNA to down-regulate Arnt protein expression revealed that TCDD-induced G(1) arrest is absolutely dependent on the Arnt protein.

The aryl hydrocarbon receptor predisposes hepatocytes to Fas-mediated apoptosis.

Liver homeostasis is achieved by the removal of diseased and damaged hepatocytes and their coordinated replacement to maintain a constant liver cell mass. Cirrhosis, viral hepatitis, and toxic drug effects can all trigger apoptosis in the liver as a means of removing the unwanted cells, and the Fas "death receptor" pathway comprises a major physiological mechanism by which this occurs. The susceptibility to Fas-mediated apoptosis is, in part, a function of the hepatocyte's proteome. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known to influence apoptosis, conceivably by regulating the expression of genes involved in apoptotic signaling. In this article, we present evidence demonstrating that AhR expression and function promote apoptosis in liver cells in response to Fas stimulation. Reintroduction of the AhR into the AhR-negative BP8 hepatoma cells as well as into primary hepatocytes from AhR knockout mice increases the magnitude of cell death in response to Fas ligand. Enhanced apoptosis correlates with increased caspase activity and mitochondrial cytochrome c release but not with the expression of several Bcl-2 family proteins. In vivo studies showed that in contrast to wild-type mice, AhR knockout mice are protected from the lethal effects of the anti-Fas Jo2 antibody. Moreover, down-regulation of the aryl hydrocarbon receptor nuclear translocator protein in vivo by adenovirus-mediated RNA interference to suppress AhR activity provided wild-type mice partial protection from Jo2-induced lethality.

Cytochrome P4501A1 promotes G1 phase cell cycle progression by controlling aryl hydrocarbon receptor activity.

The aryl hydrocarbon receptor (AhR) transcription factor is increasingly recognized as functioning in cell cycle control. Several recent reports have shown that AhR activity in the absence of exogenous agonists or presence of the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin can affect G1 phase progression in cultured cells. Serum release of serum-starved (G0) 5L rat hepatoma cells triggers transient AhR activation and P4501A1 protein expression concomitant with the G0/G1-to-S phase transition. In contrast, sustained AhR activation in response to TCDD treatment increases p27Kip1 expression in addition to P4501A1, resulting in G1 phase cell cycle arrest. Treating serum-released 5L cells with the alkyne metabolism-based P4501A1 inhibitor 1-(1-propynyl)pyrene results in prolonged AhR activation, enhanced p27Kip1 expression, and G1 phase arrest after serum release. The data are consistent with a cell cycle role for P4501A1 because they show that P4501A1 negatively regulates the duration of AhR action through the metabolic removal of the receptor agonist, thereby preventing AhR-mediated G1 phase arrest.

Aryl hydrocarbon receptor-mediated cell cycle control.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor responsive to both natural and man-made environmental compounds. AhR-mediated changes in gene expression frequently affect cell growth, and recent evidence reveals a direct role for the AhR in cell cycle control. This review examines the functional interaction between the AhR and the retinoblastoma tumor suppressor protein (pRb), and its impact on the G1 phase of the cell cycle. The discussion emphasizes gaps in our mechanistic understanding, and reveals the AhR signaling pathway as a novel drug target to control cell proliferation.