Search

Viral-mediated in vivo gene delivery methods currently dominate among therapeutic strategies within the clinical and experimental settings, albeit with well documented limitations arising from immunologic constraints. In this study, we demonstrate the utility of nonviral hepatotropic in vivo gene delivery of unpackaged expression constructs, including one encoding fibroblast growth factor 21 (FGF21). FGF21 is an important hepatokine whose expression positively correlates with therapeutic outcomes across various animal models of obesity. Our data demonstrate that FGF21 expression can be restored into the livers of immunocompetent FGF21 knockout mice for at least 2 weeks after a single injection with an FGF21 expression plasmid. In wild-type C57BL6/J mice, in vivo transfection with an FGF21-expressing plasmid induced weight loss, decreased adiposity, and activated thermogenesis in white fat within 2 weeks. Furthermore, in vivo FGF21 gene delivery protected C57BL6/J mice against diet-induced obesity by decreasing adiposity and increasing uncoupling protein 1-dependent thermogenesis in brown fat and by boosting respiratory capacity in subcutaneous and perigonadal white fat. Together, the data illustrate a facile and effective methodology for delivering prolonged protein expression specifically to the liver. We contend that this method will find utility in basic science research as a practical means to enhance in vivo studies characterizing liver protein function. We further believe our data provide a rationale for further exploring the potential clinical utility of nonviral gene therapy in mouse models of disease. SIGNIFICANCE STATEMENT: This study presents a valuable method for nonviral gene delivery in mice that improves upon existing techniques. The data provide a rationale for further exploring the potential clinical utility of nonviral gene therapy in mouse models of disease and will likely enhance in vivo studies characterizing liver protein function.

In the wake of the

This article provides a description of the rationale and processes adopted by the Gulf Coast Health Alliance: Health Risks related to the Macondo Spill consortium to evaluate and communicate the risk of exposure to polycyclic aromatic hydrocarbons (PAHs) in seafood over several years following the Deepwater Horizon disaster and subsequent oil spill. We examined gaps in knowledge associated with PAH toxicity following exposure to petrogenic (oil-derived) PAHs by studying the metabolic fate of PAHs and their potential toxicity using sophisticated analytical methods. Using the data generated, we developed a risk communication strategy designed to meet the needs of the stakeholder communities including a consumption guideline calculator, a web-based tool to reconcile seafood consumption with risk of adverse health effects.

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.

The aryl hydrocarbon receptor (AHR) has been studied for over 40 years, yet our understanding of this ligand-activated transcription factor remains incomplete. Each year, novel findings continually force us to rethink the role of the AHR in mammalian biology. The AHR has historically been studied within the context of potent activation via AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), with a focus on how the AHR mediates TCDD toxicity. Research has subsequently revealed that the AHR is actively involved in distinct physiological processes ranging from the development of the liver and reproductive organs, to immune system function and wound healing. More recently, the AHR was implicated in the regulation of energy metabolism and is currently being investigated as a potential therapeutic target for obesity. In this review, we re-trace the steps through which the early toxicological studies of TCDD led to the conceptual framework for the AHR as a potential therapeutic target in metabolic disease. We additionally discuss the key discoveries that have been made concerning the role of the AHR in energy metabolism, as well as the current and future directions of the field.

Fibroblast growth factor 21 (FGF21) is an important liver-secreted hormone that activates thermogenesis in white and brown fat deposits. In various models of obesity, FGF21 administration consistently facilitates weight loss and improved metabolic function. Several FGF21 variants, which have been engineered to improve protein stability and solubility in solutions containing preservatives, are currently in human clinical trials. In addition, in vivo FGF21 gene therapy using viral vector is being explored as an alternative therapeutic approach. In this study, we present a simpler method of in vivo FGF21 gene therapy, in which liver-specific delivery of an unpackaged plasmid construct expressing an HA-tagged FGF21 protein increases de novo hepatic FGF21 production and secretion in mice. Our data show that FGF21 protein expression can be successfully restored into the livers of FGF21 conditional knockout mice for at least two weeks after a single tail vein injection with the expression plasmid, and that the HA-tagged protein is secreted and readily detectable in serum. In wild-type C57BL6/J mice, in vivo plasmid delivery significantly increased hepatic FGF21 protein 2.3-fold after two weeks, and was associated with reduced body mass and a 14% reduction in fasting serum glucose. In addition, elevated hepatic FGF21 levels correlated with a 27% decrease in the ratio of fat to body mass, visibly smaller subcutaneous and visceral white fat adipocytes, and a 3.3-fold increase in uncoupling protein 1-dependent mitochondrial respiration in the white fat. Together, these data suggest that in vivo plasmid delivery may potentially be an effective strategy for promoting hepatic FGF21 expression in models of obesity. We are currently testing this hypothesis with experiments in high-fat diet-challenged mice.

The cycling and fate of polycyclic aromatic hydrocarbons (PAHs) is not well understood in estuarine systems. It is critical now more than ever given the increased ecosystem pressures on these critical coastal habitats. A budget of PAHs and cycling has been created for Galveston Bay (Texas) in the northwestern Gulf of Mexico, an estuary surrounded by 30–50% of the US capacity of oil refineries and chemical industry. We estimate that approximately 3 to 4 mt per year of pyrogenic PAHs are introduced to Galveston Bay via gaseous exchange from the atmosphere (ca. 2 mt/year) in addition to numerous spills of petrogenic PAHs from oil and gas operations (ca. 1.0 to 1.9 mt/year). PAHs are cycled through and stored in the biota, and ca. 20 to 30% of the total (0.8 to 1.5 mt per year) are estimated to be buried in the sediments. Oysters concentrate PAHs to levels above their surroundings (water and sediments) and contain substantially greater concentrations than other fish catch (shrimp, blue crabs and fin fish). Smaller organisms (infaunal invertebrates, phytoplankton and zooplankton) might also retain a significant fraction of the total, but direct evidence for this is lacking. The amount of PAHs delivered to humans in seafood, based on reported landings, is trivially small compared to the total inputs, sediment accumulation and other possible fates (metabolic remineralization, export in tides, etc.), which remain poorly known. The generally higher concentrations in biota from Galveston Bay compared to other coastal habitats can be attributed to both intermittent spills of gas and oil and the bay's close proximity to high production of pyrogenic PAHs within the urban industrial complex of the city of Houston as well as periodic flood events that transport PAHs from land surfaces to the Bay.

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.

Decades of research on the Aryl hydrocarbon Receptor (AhR) has unveiled its involvement in the toxicity of halogenated and polycyclic aromatic hydrocarbons, and a myriad of normal physiological processes. The molecular dissection of AhR biology has centered on a canonical signaling pathway in an effort to mechanistically reconcile the diverse pathophysiological effects of exposure to environmental pollutants. As a consequence, we now know that canonical signaling can explain many but not all of the AhR-mediated effects. Here we describe recent findings that point to non-canonical signaling pathways, and focus on a novel AhR interaction with the Krüppel-like Factor 6 protein responsible for previously un-recognized epigenetic changes in the chromatin affecting gene expression.

A community-based participatory research was utilized to address the coastal community's concern regarding Deepwater Horizon oil contamination of seafood. Therefore, we analyzed polycyclic aromatic hydrocarbons (PAHs), major toxic constituents of crude oil, in the seafood collected from gulf coast (Louisiana, Alabama and Mississippi) during December 2011-February 2014. PAHs were extracted from edible part of shrimp, oysters, and crabs by the QuEChERS/dsPE procedure and analyzed by gas chromatography-mass spectrometry. The total PAHs data were further analyzed using the General Linear Mixed Model procedure of the SAS (Version 9.3, SAS Institute, Inc., Cary, NC) statistical software. Brown shrimp showed statistically significant differences in PAHs levels with respect to time and locations while white shrimp showed differences at various time points. PAHs levels in oyster and crab samples were not statistically different at the Type I error of 0.05. Overall, the PAHs levels are far below FDA levels of concern for human consumption.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor highly expressed in hepatocytes. Researchers have employed global and liver-specific conditional Ahr knockout mouse models to characterize the physiological roles of the AHR, however, the gestational timing of AHR loss in these models can complicate efforts to distinguish the direct and indirect effects of post-gestational AHR deficiency. Utilizing a novel tamoxifen-inducible AHR knockout mouse model, we analyzed the effects of hepatocyte-targeted AHR loss in adult mice. The data demonstrate that AHR deficiency significantly reduces weight gain and adiposity, and increases multilocular lipid droplet formation within perigonadal white adipose tissue (gWAT). Protein and mRNA expression of fibroblast growth factor 21 (FGF21), an important hepatokine that activates thermogenesis in brown adipose tissue (BAT) and gWAT, significantly increases upon AHR loss and correlates with a significant increase of BAT and gWAT respiratory capacity. Confirming the role of FGF21 in mediating these effects, this phenotype is reversed in mice concomitantly lacking AHR and FGF21 expression. Chromatin immunoprecipitation analyses suggest that the AHR may constitutively suppress Fgf21 transcription through binding to a newly identified xenobiotic response element within the Fgf21 promoter. The data demonstrate an important AHR-FGF21 regulatory axis that influences adipose biology and may represent a &ldquo, druggable&rdquo, therapeutic target for obesity and its related metabolic disorders.

IMPACT: The Bench Tutorials Program is an independent study course in biomedical research in which high school students are paired with graduate and post-doctoral students during the academic year. The purpose is to enhance the rigor of high school science education and build the pipeline of tomorrow’s researchers. OBJECTIVES/GOALS: The Bench Tutorials Program: ο Proficiency in research design, implementation, and presentation; ο Acquisition of hands-on laboratory skills; ο Increase in scientific literacy; ο Increase in analytical skills and critical thinking; ο Career in science; ο Build the pipeline of tomorrow’s biomedical researchers METHODS/STUDY POPULATION: High School seniors are paired with graduate and postdoc mentors through a matching process. Students spend approximately four hours/week in supervised instruction and research from a participating laboratory in addition to classroom experience at their High School. Mentors design research projects relating to the larger research framework of their laboratories. In light of COVID-19, approaches have been adjusted to maintain the program safely through a hybrid method of using the high school lab for hands-on learning and through the use of Go-Pros ’s to enable our mentors to video and narrate as they conduct experiments in their own labs to teach their mentees scientific methods and processes. RESULTS/ANTICIPATED RESULTS: Since inception, more than 400 students and mentors have participated in the Bench Tutorial’s program. This year we found a way to continue the program under COVID-19 restraints without putting anyone in harms way. Go-Pros have been essential for our program to maintain continuity for high school students who receive academic credit for this course. This program is also one of few in which our graduate students have the opportunity to serve as mentors in the scientific setting. Using Go-Pro’s will also enable us to provide teaching videos online for other academic institutions, so even in the absence of COVID-19 in the future, the continued use of these devices will still be of great value. DISCUSSION/SIGNIFICANCE OF FINDINGS: High school students are afforded the ability to work on cutting edge research projects alongside graduate students and postdocs, who are afforded the chance to mentor and teach. Due to the COVID-19 pandemic, we have successfully adjusted our methods for teaching through the use of Go-Pro technology.

Background The association between proximity to oil refineries and cancer rate is largely unknown. We sought to compare the rate of cancer (bladder, breast, colon, lung, lymphoma, and prostate) according to proximity to an oil refinery in Texas. Methods A total of 6 302 265 persons aged 20 years or older resided within 30 miles of an oil refinery from 2010 to 2014. We used multilevel zero-inflated Poisson regression models to examine the association between proximity to an oil refinery and cancer rate. Results We observed that proximity to an oil refinery was associated with a statistically significantly increased risk of incident cancer diagnosis across all cancer types. For example, persons residing within 0-10 (risk ratio [RR] = 1.13, 95% confidence interval [CI] = 1.07 to 1.19) and 11-20 (RR = 1.05, 95% CI = 1.00 to 1.11) miles were statistically significantly more likely to be diagnosed with lymphoma than individuals who lived within 21-30 miles of an oil refinery. We also observed differences in stage of cancer at diagnosis according to proximity to an oil refinery. Moreover, persons residing within 0-10 miles were more likely to be diagnosed with distant metastasis and/or systemic disease than people residing 21-30 miles from an oil refinery. The greatest risk of distant disease was observed in patients diagnosed with bladder cancer living within 0-10 vs 21-30 miles (RR = 1.30, 95% CI = 1.02 to 1.65), respectively. Conclusions Proximity to an oil refinery was associated with an increased risk of multiple cancer types. We also observed statistically significantly increased risk of regional and distant/metastatic disease according to proximity to an oil refinery.

This paper is intended to complement our extended documentation and analysis of the activities of the Gulf Coast Health Alliance: Health Risks related to the Macondo Spill project Community Outreach and Dissemination Core entitled, “Building and maintaining a citizen science network with fishermen and fishing communities after the Deepwater Horizon oil disaster using a Community-Based Participatory Research (CBPR) approach.” We discuss nuances of CBPR practice, including trust-building, clarification of stakeholder expectations, balancing timelines and agendas, cultural fluency, and the importance of regional history—political-economic context, regulatory practices, and cultural life-ways—in creating social dynamics that overarch and underpin the entire process. We examine the unique role of knowledge-making hybrid structures like the project’s Fishermen’s citizen science network and compare/contrast this structure with other models of participatory science or deliberation. Finally, we reiterate the importance of environmental health literacy efforts, summarize project outcomes, and offer thoughts on the future roles of collaborative efforts among communities and institutional science in environmental public health.

When the Deepwater Horizon oil rig blew out in 2010, the immediate threats to productive deep water and estuarial fisheries and the region’s fishing and energy economies were obvious. Less immediately obvious, but equally unsettling, were risks to human health posed by potential damage to the regional food web. This paper describes grassroots and regional efforts by the Gulf Coast Health Alliance: health risks related to the Macondo Spill Fishermen’s Citizen Science Network project. Using a community-based participatory research approach and a citizen science structure, the multiyear project measured exposure to petrogenic polycyclic aromatic hydrocarbons, researched the toxicity of these polycyclic aromatic hydrocarbon compounds, and communicated project findings and seafood consumption guidelines throughout the region (coastal Louisiana, Mississippi, and Alabama). Description/analysis focuses primarily on the process of building a network of working fishermen and developing group environmental health literacy competencies.

The aryl hydrocarbon receptor (AhR) is a cytosolic ligand-activated transcription factor historically known for its role in xenobiotic metabolism. Although AhR activity has previously been shown to play a cytoprotective role against intrinsic apoptotic stimuli, the underlying mechanism by which AhR confers cytoprotection against apoptosis is largely unknown. Here, we demonstrate that activation of AhR by the tryptophan catabolite cinnabarinic acid (CA) directly upregulates expression of stanniocalcin 2 (Stc2) to elicit cytoprotection against apoptosis induced by endoplasmic reticulum stress and oxidative stress. Chromatin immunoprecipitation studies demonstrated that CA treatment induces direct AhR binding to a region of the Stc2 promoter containing multiple xenobiotic response elements. Using isolated primary hepatocytes from AhR wild-type (AhR floxed) and liver-specific AhR conditional knockout mice, we showed that pretreatment with CA conferred cytoprotection against hydrogen peroxide (H(2)O(2))-, thapsigargin-, and ethanol-induced apoptosis in an AhR-dependent manner. Furthermore, suppressing Stc2 expression using RNA interference confirmed that the cytoprotective properties of CA against H(2)O(2), thapsigargin, and ethanol injury were absolutely dependent on Stc2. Immunochemistry revealed the presence of Stc2 in the endoplasmic reticulum and on the cell surface, consistent with Stc2 secretion and autocrine and/or paracrine signaling. Finally, in vivo data using a mouse model of acute alcohol hepatotoxicity demonstrated that CA provided cytoprotection against ethanol-induced apoptosis, hepatic microvesicular steatosis, and liver injury. Collectively, our data uncovered a novel mechanism for AhR-mediated cytoprotection in the liver that is dependent on CA-induced Stc2 activity.

The Deepwater Horizon (DWH) explosion in 2010 is the largest oil spill (Macondo) in U.S. history. We focused on gaining an understanding of the physical health and mental health effects attributable to the Macondo oil spill. This is a report of a cross-sectional cohort study (wave 1) to establish ‘baseline’ findings and meant to provide descriptive information to be used for a multi-wave, longitudinal study. Gulf Coast Health Alliance: health Risks related to the Macondo Spill (GC-HARMS) uses a Community-Based Participatory Research approach, thus including multi-disciplinary, multi-institutional academic partners and representatives of three communities impacted by the spill. Three research sites were selected for human sampling along the Gulf of Mexico coast including two from Mississippi and one from Louisiana, with Galveston, Texas, serving as a comparison site, given that it was not directly impacted by the spill. One hundred participants were selected from each community, representing adults, seniors and children, with approximately equal numbers of males and females in each group. Participants completed initial assessments including completion of a ‘baseline’ survey and, rigorous physical assessments. Results from wave 1 data collection reported herein reveal changes in self-reported physical health and mental health status following the oil spill, disparities in access to healthcare, and associations between mental health and emotional conditions related to displacement/unemployment. Few environmental health studies have been conducted in communities impacted by significant oil spills. Results imply potential prolonged effects on mental health and community vulnerability.

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.

The aryl hydrocarbon receptor (AhR), a regulator of xenobiotic toxicity, is a member of the eukaryotic Per-Arnt-Sim domain protein family of transcription factors. Recent evidence identified a novel AhR DNA recognition sequence called the nonconsensus xenobiotic response element (NC-XRE). AhR binding to the NC-XRE in response to activation by the canonical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin resulted in concomitant recruitment of carbamoyl phosphate synthase 1 (CPS1) to the NC-XRE. Studies presented here demonstrate that CPS1 is a bona fide nuclear protein involved in homocitrullination (hcit), including a key lysine residue on histone H1 (H1K34hcit). H1K34hcit represents a hitherto unknown epigenetic mark implicated in enhanced gene expression of the peptidylarginine deiminase 2 gene, itself a chromatin-modifying protein. Collectively, our data suggest that AhR activation promotes CPS1 recruitment to DNA enhancer sites in the genome, resulting in a specific enzyme-independent post-translational modification of the linker histone H1 protein (H1K34hcit), pivotal in altering local chromatin structure and transcriptional activation.

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.

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 G 1 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 μg/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-α, which prime quiescent hepatocytes to enter G 1 phase. In fact, administration of TCDD 12 h after PH, a period well beyond the priming phase, still induced the G 1 arrest. Decreased proliferation in TCDD-treated mice correlated with reduced cyclin-dependent kinase-2 (CDK2) activity, a pivotal regulator of G 1 /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 p21 Cip1 or p27 Kip1 . 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.

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.

Traditionally, the aryl hydrocarbon receptor (AHR) is considered to be a ligand-activated receptor and transcription factor responsible for the induction of drug-metabolizing enzymes. Its role in the combinatorial matrix of cell functions was neatly established long before the first report of an AHR cDNA sequence was published. Only recently, other functions of this protein have begun to be recognized. This review addresses novel findings relating to AHR functions that have resulted from experimental approaches markedly outside traditional receptor analyses. Here we examine the aspects of AHR biology relevant to its role in cell cycle regulation, from the activation of mitogen-activated protein kinases to the cross-talk between AHR and the RAS pathway and the functional significance of the interaction between AHR and the retinoblastoma protein. We have attempted to provide the reader with a balanced interpretation of the evidence, highlighting areas of consensus as well as areas still being contested.

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 aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor in eukaryotic cells that alters gene expression in response to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In 5L hepatoma cells, TCDD induces a G1 cell cycle arrest through a mechanism that involves the AhR. The retinoblastoma tumor suppressor protein (pRb) controls cell cycle progression through G1 in addition to promoting differentiation. We examined whether the human AhR or its dimerization partner, the AhR nuclear translocator, interacts with pRb as a basis of the TCDD-induced cell cycle arrest. In vivo and in vitro assays reveal a direct interaction between pRb and the AhR but not the AhR nuclear translocator protein. Binding between the AhR and pRb occurs through two distinct regions in the AhR. A high affinity site lies within the N-terminal 364 amino acids of the AhR, whereas a lower affinity binding region colocalizes with the glutamine-rich transactivation domain of the receptor. AhR ligand binding is not required for the pRb interaction per se, although immunoprecipitation experiments in 5L cells reveal that pRb associates preferentially with the liganded AhR, consistent with a requirement for ligand-induced nuclear translocation. These observations provide a mechanistic insight into AhR-mediated cell cycle arrest and a new perspective on TCDD-induced toxicity.

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.

Background: Polycyclic aromatic hydrocarbons (PAHs) are abundant and widespread environmental chemicals. They are produced naturally and through man-made processes, and they are common in organic media, including petroleum. Several PAHs are toxic, and a subset exhibit carcinogenic activity. PAHs represent a range of chemical structures based on two or more benzene rings and, depending on their source, can exhibit a variety of side modifications resulting from oxygenation, nitrogenation, and alkylation. Objectives: Here we discuss the increasing ability of contemporary analytical methods to distinguish not only different chemical structures among PAHs but also their concentrations in environmental media. Using seafood contamination following the Deepwater Horizon accident as an example, we identify issues that are emerging in the PAH risk assessment process because of increasing analytical sensitivity for individual PAHs, and we describe the paucity of toxicological literature for many of these compounds. Discussion: PAHs, including the large variety of chemically modified or substituted PAHs, are naturally occurring and may constitute health risks if human populations are exposed to hazardous levels. However, toxicity evaluations have not kept pace with modern analytic methods and their increased ability to detect substituted PAHs. Therefore, although it is possible to measure these compounds in seafood and other media, we do not have sufficient information on the potential toxicity of these compounds to incorporate them into human health risk assessments and characterizations. Conclusions: Future research efforts should strategically attempt to fill this toxicological knowledge gap so human health risk assessments of PAHs in environmental media or food can be better determined. This is especially important in the aftermath of petroleum spills. Citation: Wickliffe J, Overton E, Frickel S, Howard J, Wilson M, Simon B, Echsner S, Nguyen D, Gauthe D, Blake D, Miller C, Elferink C, Ansari S, Fernando H, Trapido E, Kane A. 2014. Evaluation of polycyclic aromatic hydrocarbons using analytical methods, toxicology, and risk assessment research: seafood safety after a petroleum spill as an example. Environ Health Perspect 122:6–9; http://dx.doi.org/10.1289/ehp.1306724

Proper hepatocyte function is vital for survival; thus, unrepaired destruction of the parenchymal tissue leading to liver decompensation is devastating. Therefore, understanding the homeostatic process regulating liver regeneration is clinically important, and evidence that the aryl hydrocarbon receptor (AhR) can promote cell survival after intrinsic apoptotic stimuli is integral to the regenerative process. The current study uses primary hepatocytes to identify survival mechanisms consistent with normal AhR biology. Taking advantage of the Cre-lox system to manipulate AhR status, we designed a comprehensive microarray analysis to identify immediate and direct changes in the transcriptome concomitant with the loss of the AhR. As a result, we identified a unique data set with minimal overlap, compared with previous array studies, culminating in the identification of Stanniocalcin 2 (Stc2) as a novel receptor target gene previously reported to have a cytoprotective role in endoplasmic reticulum stress. The Stc2 promoter contains multiple putative xenobiotic response elements clustered in a 250-bp region that was shown to recruit the AhR by chromatin immunoprecipitation. Of interest, Stc2 gene expression is refractory to classic exogenous AhR agonists, but responds to cellular stress in an AhR-dependent mechanism consistent with a process promoting cell survival.

The aryl hydrocarbon receptor (AhR) is a mediator of xenobiotic toxicity, best recognized for conveying the deleterious effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. The AhR functions as a ligand-activated transcription factor that binds to a canonical xenobiotic response element (XRE) in association with the heterodimerization partner, the AhR nuclear translocator (Arnt) protein. However, within the repertoire of AhR target genes identified in recent years, many lack a clearly defined XRE highlighting the growing realization that AhR-mediated gene expression seems to involve additional mechanisms distinct from the well characterized process involving the XRE. The present study characterized a novel nonconsensus XRE (NC-XRE) in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene that recruits a novel protein-DNA complex responsible for TCDD-inducible expression. DNA binding studies and reporter assays identified key residues in the NC-XRE necessary for protein-DNA binding and function, respectively. Functional studies with AhR expression constructs confirm that TCDD-inducibility is AhR-dependent and requires direct AhR-DNA binding to the NC-XRE. Chromatin immunoprecipitation and RNA interference studies reveal that the Arnt protein is not a component of the NC-XRE-bound AhR complex, suggesting that in contrast to the XRE, AhR-dependent gene expression mediated through the NC-XRE may involve a new DNA binding partner.

The aryl hydrocarbon receptor (AhR) was implicated as a mediator of xenobiotic toxicity over three decades ago. Although a complete picture continues to elude us, investigations by many laboratories during the ensuing period have revealed much about AhR biology in normal physiological processes, as well as the toxicities induced by the dioxins and related polychlorinated aromatic hydrocarbons. The findings are captured in numerous excellent reviews. This commentary attempts to inject a new perspective on some new as well as frequently overlooked observations in the context of established receptor properties. Specifically, we examine the impact of transient versus sustained receptor activation on AhR biology, and explore the potential role for cytochrome P450 expression in regulating AhR activity amongst various tissues. The growing recognition that AhR action functions through multiple mechanisms serves to further highlight the importance of limiting prolonged receptor activation.

The Ah receptor in rat hepatic cytosol was transformed to a DNA-binding form by incubation in vitro with the ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin. The transformed receptor was covalently cross-linked to a bromodeoxyuridine-substituted DNA recognition motif by exposure to ultraviolet irradiation. Analyses of the cross-linked protein-DNA complexes by gel electrophoresis and autoradiography imply that the DNA-binding form of the liganded Ah receptor is composed of two protein components, whose molecular masses are about 110 and 100 kDa. Protease digestion studies suggest that the two components have different primary structures. Photoaffinity labeling studies imply that the smaller protein is the ligand-binding component of the receptor. These findings constitute biochemical evidence that the DNA-binding form of the Ah receptor is a heterodimer.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin produces its biological effects by binding to an intracellular protein, the Ah receptor. The liganded receptor activates transcription by binding to a specific recognition motif within a dioxin-responsive enhancer upstream of the target CYP1A1 gene. Here, we have used gel retardation to analyze the interaction between the liganded Ah receptor and five circularly permuted DNA fragments that contain a receptor recognition motif. Our findings indicate that the binding of the liganded receptor to its recognition motif bends the DNA at (or near) the site of the protein-DNA interaction. This observation implies that Ah receptor-induced DNA distortion may contribute to the activation of CYP1A1 transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

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.

Background: Scanning cytometry now has many of the features (and power) of multiparameter flow cytometry while keeping its own advantages as an imaging technology. Modern instruments combine capabilities of scanning cytometry with the ability to manipulate cells. A new technology, called LEAP™ (laser-enabled analysis and processing), offers a unique combination of capabilities in cell purification and selective macromolecule delivery (optoinjection). Methods: LEAP-mediated cell purification and optoinjection effects were assessed in model experiments using adherent and suspension cell types and cell mixtures plated and processed at different densities. Optoinjection effects were visualized by delivering fluorescent dextrans into cells. Results were analyzed using the LEAP instrument's own imaging system as well as by fluorescence and confocal microscopy. Results: Live cell samples (adherent and suspension) could be purified to 90–100% purity with 50–90% yield, causing minimal cell damage depending on the cell type and plating density. Nearly one hundred percent of the targeted cells of all cell types examined could be successfully optoinjected with dextrans of 3–70 kDa, causing no visual damage to the cells. Indirect optoinjection effects were observed on untargeted cells within 5–60 μm to targeted areas under conditions used here. Conclusions: LEAP provides solutions in cell purification and targeted macromolecule delivery for traditional and challenging applications where other methods fall short. © 2006 International Society for Analytical Cytology

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 hepatocyte9s 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.

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.

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.

Proliferative breast disease (PBD) may increase a woman's risk of developing breast cancer, perhaps by decreasing cellular sensitivity to apoptosis. To determine whether resistance to apoptosis develops during PBD, we investigated apoptosis initiated through the Fas pathway in a series of cell lines that recapitulates the morphologic changes of PBD in nude/beige mice.The series of cell lines used was MCF-10A cells (parental preneoplastic human breast epithelial cells), MCF-10AT cells (transformed with T(24) Ha-ras), and MCF-10ATG3B cells (derivative cells that progress to carcinoma). Fas-mediated apoptosis, induced when a Fas monoclonal antibody bound to and activated the Fas receptor on these cells, was assessed morphologically and by flow cytometry. Levels of proteins involved in Fas-mediated apoptosis and cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP), an end product of caspase activation, were determined by immunoblotting. Bcl-2 and Bax heterodimerization was examined by coimmunoprecipitation. All statistical tests were two-sided.Sensitivity to Fas-mediated apoptosis decreased with the tumorigenic potential of cells: MCF-10A cells were extremely susceptible, MCF-10AT cells were less susceptible, and MCF-10ATG3B cells were resistant. The percentage of apoptotic cells declined, from 24% to 8% to 6%, respectively. All lines produced Fas ligand (FasL) and had comparable levels of Fas receptor, FasL, Fas-associated death-domain protein, and caspases 3 and 6. Levels of caspase 8 were similar in MCF-10A and MCF-10AT cells but about 30% lower in MCF-10ATG3B cells (P.01 but.05). Levels of caspase 10 were about 20% lower in MCF-10AT cells (P.005 but.01) and about 59% lower in MCF-10ATG3B cells than in MCF-10A cells (P.01 but.05). PARP cleavage was detected in MCF-10A and MCF-10AT cells but not in MCF-10ATG3B cells. Levels of Bax, Bid, and Bak proteins were similar in all lines, but levels of Bcl-2 were lower in MCF-10AT and MCF-10ATG3B cells than in MCF-A cells, and Bcl-2-Bax heterodimerization progressively declined in the series.Resistance to Fas-mediated apoptosis appears to develop progressively in the MCF-10AT cell series.

The aryl hydrocarbon receptor (AhR) belongs to the basic helix-loop-helix/periodicity/AhR nuclear translocator/simple-minded (Per-Arnt-Sim) family of transcription factors that regulate critical functions during development and tissue homeostasis. Within this family, the AhR is the only member conditionally activated in response to ligand binding, typified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We recently demonstrated that the AhR interacts with the retinoblastoma protein (pRb). This report presents evidence that a LXCXE motif in the AhR protein confers pRb binding, which is necessary for maximal TCDD induced G(1) arrest in rat 5L hepatoma cells. The data support a mechanism whereby pRb seems to regulate G(1) cell cycle progression distinct from the direct repression of E2F-mediated transcription. Furthermore, the results indicate that the AhR-pRb interaction regulates TCDD induction of CYP1A1, suggesting that pRb may be a general AhR coactivator.

Background: Hepatocellular carcinoma HCC)is a common complication of hepatitis C (HCV) related cirrhosis. Early diagnosis is important for the successful treatment by ablation, resection, and/or transplant. Although AFP is routinely used for screening, it is often normal or indeterminate in early cases. Therefore, a more sensitive marker of HCC would be of great interest. Protein Chip Technology (SELDI-TOF) has been used as a profiling technique for the identification of potential biomarkers which may represent proteins, protease-cleavage products, and peptides. Aim: To use SELDI-TOF to compare the serum profile of chronic HCV patients with and without HCC, and develop a biomarker panel for HCC. Methods: All patients studied had a positive HCV RNA. The patients with HCV only were enrolled in a prospective study of hepatic fibrosis markers, and were equally divided among the different stages of fibrosis. For patients with HCC all had histological verification, an AFP > 400, or fit AASLD radiographic criteria for HCC. The samples were prefractionated using Equalizer bead protein biomarker technology and SELDI ProteinChip array profiling. The fractions were analyzed on the PCS 4000 time of flight mass spectrometer using IMAC (pretreated with Cu), CM10, and Q10 chips. Data of the proteomic spectra was analyzed by Ciphergen Express data manager software with pattern track and 2-way Hierarchical Clustering algorithm. Initially, the serum of 50 patients with HCV only and 40 patients with HCC and HCV were analyzed (training set). A risk formula of peaks 1.2 fold was seen. For the validation set we used a ROC (AUROC=0.897) generated risk score cutoff of -4.11. Using this cutoff, 22/22 patients -4.11 had HCC (PPV=70%). Conclusion: We were able to develop and validate a biomarker panel utilizing 4 peaks with a MW< 20 KDa that was able to accurately separate patients with HCV only from those with HCV and HCC. Additional work to identify these putative biomarkers along with the development of assays will be necessary to evaluate their usefulness in the early detection of HCC.

The authors have employed a combination of gel retardation, protein-DNA cross-linking, and protein-protein cross-linking techniques to further examine the 2,3,7,8-tetracholorodibenzo-p-dioxin- (TCDD-) dependent changes in the Ah receptor that result in a DNA-binding conformation. Gel retardation analysis of DNA-Sepharose chromatographic fractions of rat hepatic cytosol indicated that TCDD-dependent and sequence-specific DNA binding coeluted with a 200-kDa form of the Ah receptor (peak 2) previoulsly characterized as being multimeric and having high affinity for calf thymus DNA. The TCDD-cound, 100-kDa form of the receptor (peak 1) bound weakly to the DNA recognition motif. These results indicated that the DNA-binding form of the Ah receptor is a multimer. SDS-polyacrylamide gel electrophoresis of peak 2 cross-linked to a bromodeoxyuridine-substituted DNA recognition motif indicated that this form of the receptor present in rat hepatic cytosol is composed of at least two DNA-bginding proteins of approximately 100 and 110 kDa. Using the chemical cross-linking agent dimethyl pimelimidate, they further established that the 100-kDa form of the receptor (peak 1) associates with a different protein to generate the receptor form (peak 2) that binds to the dioxin-responsive enhancer. Photoaffinity-labeling studies indicated that only the 100-kDa protein (peak 1), and not the 110-kDa protein, binds ligand. Together, thesemore » observations imply that the DNA-binding form of the Ah receptor exists as a heteromer.« less

We have reviewed the transcriptional regulation of the CYP1A1 gene, which encodes the cytochrome P450IA1 enzyme. TCDD induces CYP1A1 transcription via a receptor- and enhancer-dependent mechanism that involves multiple protein-DNA interactions. The system provides an example of enzyme regulation at the level of gene transcription.

Publisher Summary This chapter discusses the use of gel retardation to analyze protein-DNA interactions upstream of CYPIA1 gene. The gel retardation or gel mobility shift technique has become a standard tool for analyzing the interaction in vitro between DNA-binding proteins and their cognate genomic recognition sequences. The method relies on the fact that, during electrophoresis under nondenaturing conditions, protein-DNA complexes have a reduced mobility compared with protein-free DNA. Several groups have used gel retardation to analyze tetrachlorodibenzo-p-dioxin (TCDD)-inducible protein–DNA interactions at regulatory domains upstream of the CYPIA1 gene. The conditions used by each group vary somewhat, revealing some flexibility in the assay conditions. Gel retardation is also used to analyze cytosolic proteins from both mouse hepatoma cells and rodent liver. The cytosolic preparations are stored under the same conditions as the nuclear extracts, and exhibit similar stabilities.

We have measured the transcriptional gene activity of 5-aminolevulinate synthase, the first enzyme of the heme biosynthetic pathway, together with corresponding mRNA and protein levels in mouse erythroleukemic cells induced to differentiate with dimethyl sulfoxide. When the heme biosynthetic pathway was blocked by succinylacetone there was a large increase in both 5-aminolevulinate synthase activity and protein levels, and this was reversed by the addition of exogenous hemin. Transcriptional activity of the 5-aminolevulinate synthase gene and mRNA levels were both significantly increased during differentiation of cells by dimethyl sulfoxide but were not markedly altered by succinylacetone or hemin treatment. The results demonstrate that levels of 5-aminolevulinate synthase in mouse erythroleukemic cells are regulated by a significant post-transcriptional mechanism possibly at the translational level. Evidence is also presented for a less significant post-transcriptional control by heme of mRNA levels for 5-aminolevulinate synthase. These results indicate that the regulation of 5-aminolevulinate synthase in differentiating erythroid cells is complex but differs from that in liver cells where heme controls the level of 5-aminolevulinate synthase by acting primarily to inhibit gene transcription.

Reports to date have led to the conclusion that there are isozymes for 5-aminolevulinate synthase in the liver and erythroid tissue of chicken. Indeed, the existence of a multigene family for chicken 5-aminolevulinate synthase has been proposed. We find no evidence to support these proposals. In this work we show that 5-aminolevulinate synthase mRNA from chicken liver and reticulocytes is identical as determined by RNase mapping and primer extension studies and that the 5-aminolevulinate synthase protein from these tissues is the same size as judged by immunoblot analysis. We also show that a single mRNA species for 5-aminolevulinate synthase is present in chicken liver, reticulocytes, brain, and heart and an avian erythroblastosis virus-transformed chicken erythroblast cell line. Southern analysis shows the presence of only one gene copy for 5-aminolevulinate synthase in the chicken haploid genome. Overall, these results lead to the conclusion that in chickens 5-aminolevulinate synthase is encoded by a unique gene and is expressed as a single mRNA species in all tissues.

cDNA clones for rat liver 5-aminolevulinate synthase have been isolated and used to examine mRNA levels in different rat tissues. Northern hybridization analysis of total RNA from various rat tissues showed the presence of a single 5-aminolevulinate synthase mRNA species of estimated length 2.3 kilobases. Primer extension and RNase mapping studies indicated that the mRNA is identical in all tissues. Highest basal levels were seen in liver and heart. Administration of hemin to rats reduced the basal level of this mRNA only in liver but the heme precursor, 5-aminolevulinate (or its methyl ester), repressed the basal levels in liver, kidney, heart, testis, and brain. The drug 2-allyl-2-isopropylacetamide increased the mRNA level in liver and kidney only while human chorionic gonadotropin hormone elevated the level in testis. Administration of the heme precursor 5-aminolevulinate prevented these inductions. Nuclear transcriptional run-off experiments in liver cell nuclei showed that 2-allyl-2-isopropylacetamide and 5-aminolevulinate exert their effect by altering the rate of transcription of the 5-aminolevulinate synthase gene. The results indicate that a single 5-aminolevulinate synthase mRNA is expressed in all tissues and that its transcription is negatively regulated by heme.