Biological Responses of Pacific Herring Embryos to Crude Oil Are Quantifiable at Exposure Levels Below Conventional Limits of Quantitation for PAHs in Water and Tissues.

Pacific herring (Clupea pallasii), a cornerstone of marine food webs, generally spawn on marine macroalgae in shallow nearshore areas that are disproportionately at risk from oil spills. Herring embryos are also highly susceptible to toxicity from chemicals leaching from oil stranded in intertidal and subtidal zones. The water-soluble components of crude oil trigger an adverse outcome pathway that involves disruption of the physiological functions of cardiomyocytes in the embryonic herring heart. In previous studies, impaired ionoregulation (calcium and potassium cycling) in response to specific polycyclic aromatic hydrocarbons (PAHs) corresponds to lethal embryolarval heart failure or subtle chamber malformations at the high and low ends of the PAH exposure range, respectively. Sublethal cardiotoxicity, which involves an abnormal outgrowth (ballooning) of the cardiac ventricular chamber soon after hatching, subsequently compromises juvenile heart structure and function, leading to pathological hypertrophy of the ventricle and reduced individual fitness, measured as cardiorespiratory performance. Previous studies have not established a threshold for these sublethal and delayed-in-time effects, even with total (∑)PAH exposures as low as 29 ng/g of wet weight (tissue dose). Here, we extend these earlier findings showing that (1) cyp1a gene expression provides an oil exposure metric that is more sensitive than typical quantitation of PAHs via GC-MS and (2) heart morphometrics in herring embryos provide a similarly sensitive measure of toxic response. Early life stage injury to herring (impaired heart development) thus occurs below the quantitation limits for PAHs in both water and embryonic tissues as a conventional basis for assessing oil-induced losses to coastal marine ecosystems.

Low-level embryonic crude oil exposure disrupts ventricular ballooning and subsequent trabeculation in Pacific herring.

There is a growing awareness that transient, sublethal embryonic exposure to crude oils cause subtle but important forms of delayed toxicity in fish. While the precise mechanisms for this loss of individual fitness are not well understood, they involve the disruption of early cardiogenesis and a subsequent pathological remodeling of the heart much later in juveniles. This developmental cardiotoxicity is attributable, in turn, to the inhibitory actions of crude oil-derived mixtures of polycyclic aromatic compounds (PACs) on specific ion channels and other proteins that collectively drive the rhythmic contractions of heart muscle cells via excitation-contraction coupling. Here we exposed Pacific herring (Clupea pallasi) embryos to oiled gravel effluent yielding ΣPAC concentrations as low as ~ 1 µg/L (64 ng/g in tissues). Upon hatching in clean seawater, and following the depuration of tissue PACs (as evidenced by basal levels of cyp1a gene expression), the ventricles of larval herring hearts showed a concentration-dependent reduction in posterior growth (ballooning). This was followed weeks later in feeding larvae by abnormal trabeculation, or formation of the finger-like projections of interior spongy myocardium, and months later with hypertrophy (overgrowth) of the spongy myocardium in early juveniles. Given that heart muscle cell differentiation and migration are driven by Ca2+-dependent intracellular signaling, the observed disruption of ventricular morphogenesis was likely a secondary (downstream) consequence of reduced calcium cycling and contractility in embryonic cardiomyocytes. We propose defective trabeculation as a promising phenotypic anchor for novel morphometric indicators of latent cardiac injury in oil-exposed herring, including an abnormal persistence of cardiac jelly in the ventricle wall and cardiomyocyte hyperproliferation. At a corresponding molecular level, quantitative expression assays in the present study also support biomarker roles for genes known to be involved in muscle contractility (atp2a2, myl7, myh7), cardiomyocyte precursor fate (nkx2.5) and ventricular trabeculation (nrg2, and hbegfa). Overall, our findings reinforce both proximal and indirect roles for dysregulated intracellular calcium cycling in the canonical fish early life stage crude oil toxicity syndrome. More work on Ca2+-mediated cellular dynamics and transcription in developing cardiomyocytes is needed. Nevertheless, the highly specific actions of ΣPAC mixtures on the heart at low, parts-per-billion tissue concentrations directly contravene classical assumptions of baseline (i.e., non-specific) crude oil toxicity.

Overexpression of MHCII by hepatocytes in alcoholic hepatitis (AH) compared to non-alcoholic steatohepatitis (NASH) and normal controls.

Previously we have shown that in autoimmune hepatitis CD4 positive lymphocytes form an immunologic synapse with hepatocytes, leading to gradual diminishing and elimination of the hepatocyte. We wondered whether a similar mechanism may occur in alcoholic hepatitis (AH) and non-alcoholic steatohepatitis (NASH). We conducted immunofluorescence studies of expression of MHCII, the binding partner of CD4, on patient liver biopsies of AH, NASH, and normal controls. In cases of alcoholic hepatitis, there was prominent sinusoidal expression of MHC II; In NASH biopsies there was comparatively lower expression of MHC II, but still more than control tissue. Immunohistochemical stain for CD4 showed CD4 positive lymphocytes closely associated with hepatocytes in AH biopsies. Furthermore, expression levels of the multifunctional cytokine IL-1α was higher in AH compared to NASH and control biopsies. These results underlie the more severe nature of alcoholic hepatitis and underscore the autoimmune mechanisms involved in the liver damage found in alcoholic hepatitis.

Embryonic Crude Oil Exposure Impairs Growth and Lipid Allocation in a Keystone Arctic Forage Fish.

As Arctic ice recedes, future oil spills pose increasing risk to keystone species and the ecosystems they support. We show that Polar cod (Boreogadus saida), an energy-rich forage fish for marine mammals, seabirds, and other fish, are highly sensitive to developmental impacts of crude oil. Transient oil exposures ≥300 µg/L during mid-organogenesis disrupted the normal patterning of the jaw as well as the formation and function of the heart, in a manner expected to be lethal to post-hatch larvae. More importantly, we found that exposure to lower levels of oil caused a dysregulation of lipid metabolism and growth that persisted in morphologically normal juveniles. As lipid content is critical for overwinter survival and recruitment, we anticipate Polar cod losses following Arctic oil spills as a consequence of both near-term and delayed mortality. These losses will likely influence energy flow within Arctic food webs in ways that are as-yet poorly understood.

The different expression of tumor suppressors, RASSF1A, RUNX3, and GSTP1, in patients with alcoholic steatohepatitis (ASH) vs non-alcoholic steatohepatitis (NASH).

As the fifth most common cancer and the second leading cause of cancer related deaths worldwide, hepatocellular carcinoma (HCC) causes up to one million deaths annually. Alcoholic steatohepatitis (ASH) and non-alcoholic steatohepatitis (NASH) are becoming the two major risk factors because both may develop liver fibrosis and hepatocellular carcinoma (HCC) if left untreated. However, compared with 3-10% of patients with ASH may progress to HCC annually, about only 0.5% NASH patients may progress to HCC annually. The present study is to clarify the protein expression differences of tumor suppressor genes (TSGs) between ASH and NASH. In liver biopsied specimens from NASH and ASH patients, using an immunofluorescence method and morphometrically quantitating the fluorescence intensity, we studied the protein expression within hepatocytes cytoplasm of candidate TSGs including RUNX3, GSTP1, and RASSF1A. Compared with the control group of patients, the expression levels of all three proteins were upregulated in the ASH group of patients (p < .001 in all molecules). While RUNX3 was upregulated, GSTP1 and RASSF1 did not change in the NASH group of patients. The most important finding is that compared with the ASH group of patients, the expression levels of all three TSG proteins, RUNX3, GSTP1, and RASSF1, were significantly lower in the NASH group of patients (p < .001 in all three molecules). These results confirmed our previous finding that there are significant differences of many molecules including TSGs that changed in NASH compared to ASH. Thus, we conclude that there are significantly different TSGs and pathways involved during the pathogenesis of HCC development in NASH compared to ASH that may help to develop different strategies for prevention and treatment of NASH and ASH patients.

Identification of invadopodia by TKS5 staining in human cancer lines and patient tumor samples.

Invadopodia, cancer cell protrusive structures with associated proteolytic activity, provide cancer cells with the ability to remodel the extracellular matrix. Invadopodia facilitate invasive migration and their formation correlates with cancer cell invasiveness and metastatic potential. The unambiguous identification of invadopodia is an important step to undergo studies on invadopodia regulatory inputs, functional outputs, as well as the prevalence and significance of invadopodia for cancer cells and human tumors. The adaptor protein TKS5 is a known invadopodia regulatory protein, which is necessary for invadopodia formation and activity. TKS5 is highly enriched at invadopodia and, unlike other commonly used invadopodia markers, it does not accumulate significantly in other types of cellular protrusions. However, the use of TKS5 as a marker of invadopodia has not been generalized, in part due to the availability of suitable antibodies against the human protein. We have evaluated two commercial antibodies raised against human TKS5. Here, we detail protocols for the detection of invadopodia-associated TKS5 in human cells in culture and in paraffin-embedded archived tumor surgical specimens using commercial antibodies. These methods should facilitate the identification and study of human invadopodia. •TKS5 staining identifies invadopodia in human cancer cell lines and archived surgical tumor specimens.

Crude oil cardiotoxicity to red drum embryos is independent of oil dispersion energy.

The potential bioavailability of toxic chemicals from oil spills to water column organisms such as fish embryos may be influenced by physical dispersion along an energy gradient. For example, a surface slick with minimal wave action (low energy) could potentially produce different toxic effects from high energy situations such as pressurized discharge from a blown wellhead. Here we directly compared the toxicity of water accommodated fractions (WAFs) of oil prepared with low and high mixing energy (LEWAFs and HEWAFs, respectively) using surface oil samples collected during the 2010 Deepwater Horizon spill, and embryos of a representative nearshore species, red drum (Sciaenops ocellatus). Biological effects of each WAF type was quantified with several functional and morphological indices of developmental cardiotoxicity, providing additional insight into species-specific responses to oil exposure. Although the two WAF preparations yielded different profiles of polycyclic aromatic hydrocarbons (PAHs), cardiotoxic phenotypes were essentially identical. Based on benchmark thresholds for both morphological and functional cardiotoxicity, in general LEWAFs had lower thresholds for these phenotypes than HEWAFs based on total PAH measures. However, HEWAF and LEWAF toxicity thresholds were more similar when calculated based on estimates of dissolved PAHs only. Differences in thresholds were attributable to the weathering state of the oil samples.

Different roles of FAT10, FOXO1, and ADRA2A in hepatocellular carcinoma tumorigenesis in patients with alcoholic steatohepatitis (ASH) vs non-alcoholic steatohepatitis (NASH).

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the second leading cause of cancer related deaths worldwide. Among others, non-alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH) are the two major risk factors as both of them may develop cirrhosis and hepatocellular carcinoma (HCC) if left untreated. However, patients with NASH progress to HCC at a rate around 0.5% annually, while 3-10% ASH patients may progress to HCC annually. The present study is to demonstrate the molecular differences in oncogenesis pathway between NASH and ASH. By using immunofluorescence study and quantitating the fluorescence intensity morphometrically in liver biopsied specimens from NASH and ASH patients, the protein expression of candidate molecules within hepatocytes cytoplasm are studied, including two HCC-related molecules FAT10 and FOXO1, and one GPCR pathway related molecule ADRA2A. Compared with the control group patients, the expression levels of all the molecules were upregulated in the ASH group of patients (p < 0.001 in all molecules), while FAT10 and ADRA2A were upregulated, FOXO1 did not change in the NASH group of patients. The most important finding is that compared with the ASH group of patients, the expression levels of all three molecules were significantly lower than in the NASH group of patients (p < 0.001 in all molecules). These results confirmed our previous finding that there are significant differences of molecules change in ASH compared to NASH. Thus, we conclude that there are significantly different molecules and pathways involved during the pathogenesis of HCC development in ASH compared to NASH which could help explain why the tumorigenic rate is different in ASH and NASH.

Expression of proteins upregulated in hepatocellular carcinoma in patients with alcoholic hepatitis (AH) compared to non-alcoholic steatohepatitis (NASH): An immunohistochemical analysis of candidate proteins.

Both non-alcoholic steatohepatitis (NASH) and alcoholic hepatitis (AH) can lead to cirrhosis and hepatocellular carcinoma. However, the rate of progression to cirrhosis and tumorigenesis in AH is greater than that in NASH. We asked whether there are differences between the two conditions in the expression levels of proteins involved in the pathogenesis of hepatocellular carcinoma. The proteins tested were presented at the 2017 American Association for the Study of Liver Diseases (AASLD) Liver Meeting as overexpressed in hepatocellular carcinoma: KLF4, SCL19A1, FANCG, HRH-1, DNMT1, DNMT3B, TNFR2, DUSP4, EGFR, Integrin α6, HDACII, PDE3A, BCL-XL, and MTCO2. The expression of these proteins was measured in liver biopsy sections from NASH and AH patients using immunohistochemical staining with fluorescent antibodies and then quantifying the fluorescence intensity morphometrically. In AH patients, levels of all tested proteins except HRH-1 were elevated compared to normal patients. In NASH patients, KLF4, SCL19A1, FANCG, HDACII, BCL-XL levels were increased compared to normal controls while HRH-1, DNMT1 and PDE3A levels were decreased. The relative expression of all proteins studied except BCL-XL was significantly higher in AH compared to NASH. In conclusion, proteins involved in hepatocellular cancer development are more highly expressed in AH compared to NASH and normal liver, which corresponds with the higher rate of tumorigenesis in AH patients compared to NASH patients.

Alcoholic hepatitis versus non-alcoholic steatohepatitis: Levels of expression of some proteins involved in tumorigenesis.

Non-alcoholic steatohepatitis (NASH) is commonly associated with obesity, type 2 diabetes, and/or hypertriglyceridemia, while alcoholic steatohepatitis (ASH) is associated with alcohol abuse. Both NASH and ASH patients can develop cirrhosis and hepatocellular carcinoma (HCC) if left untreated. However, the rate of tumorigenesis in NASH and ASH appears to be different. Individuals with NASH progress to HCC at a rate of 0.5% annually (Lindenmeyer and McCullough, 2018), when individuals with ASH progress to HCC at a rate of 3-10% annually (Schwartz and Reinus, 2012). Thus, the objective of our study is to determine if there are differences in NASH versus ASH in the levels of different proteins expressed involved in cancer development. The method used was measuring the proteins expressed in liver biopsied sections from NASH and ASH patients using immunohistochemical staining with fluorescent antibodies and then quantitating the fluorescence intensity morphometrically. The 20 proteins tested are parts of the Ingenuity Canonical Pathway of Molecular Mechanisms of Cancer and include: RAP2B, NAIP, FYN, PAK6, SUV39H1, GNAI1, BAX, E2F3, CKDN2B, BAK1, BCL2, DIABLO, RASGRF2, GNA15, PIK3CB, BRCA1, MAP2K1, BIRC3, CDK2, and ATM. In ASH, the proteins that showed upregulated levels of expression were SUV39H1, E2F3, BCL2, BAK1, BIRC3, and GNAI1. In NASH, the proteins that showed upregulated levels of expression were BAK1 and GNAI1 and the protein that showed downregulated level of expression was BCL2. Additionally, levels of expression for SUV39H1, E2F3, BCL2, BAK1, BIRC3, and GNAI1 were significant upregulated in ASH compared to NASH. These results showed significant differences in ASH compared to normal liver, and significant differences in ASH compared to NASH. Thus, we conclude that there are more proteins involved in tumorigenesis in ASH compared to NASH and in ASH compared to normal liver, which is consistent with the known tumor development rate in ASH and NASH.

The effects of weathering and chemical dispersion on Deepwater Horizon crude oil toxicity to mahi-mahi (Coryphaena hippurus) early life stages.

To better understand the impact of the Deepwater Horizon (DWH) incident on commercially and ecologically important pelagic fish species, a mahi-mahi spawning program was developed to assess the effect of embryonic exposure to DWH crude oil with particular emphasis on the effects of weathering and dispersant on the magnitude of toxicity. Acute lethality (96 h LC50) ranged from 45.8 (28.4-63.1) µg l(-1) ΣPAH for wellhead (source) oil to 8.8 (7.4-10.3) µg l(-1) ΣPAH for samples collected from the surface slick, reinforcing previous work that weathered oil is more toxic on a ΣPAH basis. Differences in toxicity appear related to the amount of dissolved 3 ringed PAHs. The dispersant Corexit 9500 did not influence acute lethality of oil preparations. Embryonic oil exposure resulted in cardiotoxicity after 48 h, as evident from pericardial edema and reduced atrial contractility. Whereas pericardial edema appeared to correlate well with acute lethality at 96 h, atrial contractility did not. However, sub-lethal cardiotoxicity may impact long-term performance and survival. Dispersant did not affect the occurrence of pericardial edema; however, there was an apparent reduction in atrial contractility at 48 h of exposure. Pericardial edema at 48 h and lethality at 96 h were equally sensitive endpoints in mahi-mahi.

Aberrant modulation of the BRCA1 and G1/S cell cycle pathways in alcoholic hepatitis patients with Mallory Denk Bodies revealed by RNA sequencing.

Mallory-Denk Bodies (MDBs) are prevalent in various liver diseases including alcoholic hepatitis (AH) and are formed in mice livers by feeding DDC. Liver injury from alcohol administration causes balloon hepatocytes and MDB formation impeding liver regeneration. By comparing AH livers where MDBs had formed with normal liver transcriptomes obtained by RNA sequencing (RNA-Seq), there was significant upregulation of BRCA1-mediated signaling and G1/S cell cycle checkpoint pathways. The transcriptional architecture of differentially expressed genes from AH livers reflected step-wise transcriptional changes progressing to AH. Key molecules such as BRCA1, p15 and p21 were significantly upregulated both in AH livers and in the livers of the DDC re-fed mice model where MDBs had formed. The increase of G1/S cell cycle checkpoint inhibitors p15 and p21 results in cell cycle arrest and inhibition of liver regeneration, implying that p15 and p21 could be exploited for the identification of specific targets for the treatment of liver disease. Provided here for the first time is the RNA-Seq data that represents the fully annotated catalogue of the expression of mRNAs. The most prominent alterations observed were the changes in BRCA1-mediated signaling and G1/S cell cycle checkpoint pathways. These new findings expand previous and related knowledge in the search for gene changes that might be critical in the understanding of the underlying progression to the development of AH.

Corresponding morphological and molecular indicators of crude oil toxicity to the developing hearts of mahi mahi.

Crude oils from distinct geological sources worldwide are toxic to developing fish hearts. When oil spills occur in fish spawning habitats, natural resource injury assessments often rely on conventional morphometric analyses of heart form and function. The extent to which visible indicators correspond to molecular markers for cardiovascular stress is unknown for pelagic predators from the Gulf of Mexico. Here we exposed mahi (Coryphaena hippurus) embryos to field-collected crude oil samples from the 2010 Deepwater Horizon disaster. We compared visible heart defects (edema, abnormal looping, reduced contractility) to changes in expression of cardiac-specific genes that are diagnostic of heart failure in humans or associated with loss-of-function zebrafish cardiac mutants. Mahi exposed to crude oil during embryogenesis displayed typical symptoms of cardiogenic syndrome as larvae. Contractility, looping, and circulatory defects were evident, but larval mahi did not exhibit downstream craniofacial and body axis abnormalities. A gradation of oil exposures yielded concentration-responsive changes in morphometric and molecular responses, with relative sensitivity being influenced by age. Our findings suggest that 1) morphometric analyses of cardiac function are more sensitive to proximal effects of crude oil-derived chemicals on the developing heart, and 2) molecular indicators reveal a longer-term adverse shift in cardiogenesis trajectory.

Altered regulation of miR-34a and miR-483-3p in alcoholic hepatitis and DDC fed mice.

MicroRNAs are small noncoding RNAs that negatively regulate gene expression by binding to the untranslated regions of their target mRNAs. Deregulation of miRNAs is shown to play pivotal roles in tumorigenesis and progression. Mallory-Denk Bodies (MDBs) are prevalent in various liver diseases including alcoholic hepatitis (AH) and are formed in mice livers by feeding DDC. By comparing AH livers where MDBs had formed with normal livers, there were significant changes of miR-34a and miR-483-3p by RNA sequencing (RNA-Seq) analyses. Real-time PCR further shows a 3- and 6-fold upregulation (respectively) of miR-34a in the AH livers and in the livers of DDC re-fed mice, while miR-483-3p was significantly downregulated in AH and DDC re-fed mice livers. This indicates that miR-34a and miR-483-3p may be crucial for liver MDB formation. P53 mRNA was found to be significantly downregulated both in the AH livers and in the livers of DDC re-fed mice, indicating that the upregulation of miR-34a is permitted by the decrease of p53 in AH since miR-34a is a main target of p53. Overexpression of miR-34a leads to an increase of p53 targets such as p27, which inhibits the cell cycle leading to cell cycle arrest. Importantly, BRCA1 is a target gene of miR-483-3p by RNA-Seq analyses and the downregulation of miR-483-3p may be the mechanism for liver MDB formation since the BRCA1 signal was markedly upregulated in AH livers. These results constitute a demonstration of the altered regulation of miR-34a and miR-483-3p in the livers of AH and mice fed DDC where MDBs formed, providing further insight into the mechanism of MDB formation mediated by miR-34a and miR-483-3p in AH.

IL-8 signaling is up-regulated in alcoholic hepatitis and DDC fed mice with Mallory Denk Bodies (MDBs) present.

Chemokines and their receptors are involved in oncogenesis and in tumor progression, invasion, and metastasis. Various chemokines also promote cell proliferation and resistance to apoptosis of stressed cells. The chemokine CXCL8, also known as interleukin-8 (IL-8), is a proinflammatory molecule that has functions within the tumor microenvironment. Deregulation of IL-8 signaling is shown to play pivotal roles in tumorigenesis and progression. Mallory-Denk Bodies (MDBs) are prevalent in various liver diseases including alcoholic hepatitis (AH) and are formed in mice livers by feeding DDC. By comparing AH livers where MDBs had formed with normal livers, there were significant changes of IL-8 signaling by RNA sequencing (RNA-Seq) analyses. Real-time PCR analysis of CXCR2 further shows a 6-fold up-regulation in AH livers and a 26-fold up-regulation in the livers of DDC re-fed mice. IL-8 mRNA was also significantly up-regulated in AH livers and DDC re-fed mice livers. This indicates that CXCR2 and IL-8 may be crucial for liver MDB formation. MDB containing balloon hepatocytes in AH livers had increased intensity of staining of the cytoplasm for both CXCR2 and IL-8. Overexpression of IL-8 leads to an increase of the mitogen activated protein kinase (MAPK) cascade and exacerbates the inflammatory cycle. These observations constitute a demonstration of the altered regulation of IL-8 signaling in the livers of AH and mice fed DDC where MDBs formed, providing further insight into the mechanism of MDB formation mediated by IL-8 signaling in AH.

Mallory-Denk Body (MDB) formation modulates Ufmylation expression epigenetically in alcoholic hepatitis (AH) and non-alcoholic steatohepatitis (NASH).

Promoter CpG island hypermethylation is an important mechanism for inactivating key cellular enzymes that mediate epigenetic processes in hepatitis-related hepatocellular carcinoma (HCC). The ubiquitin-fold modifier 1 (Ufm1) conjugation pathway (Ufmylation) plays an essential role in protein degradation, protein quality control and signal transduction. Previous studies showed that the Ufmylation pathway was downregulated in alcoholic hepatitis (AH), non-alcoholic steatohepatitis (NASH) and in mice fed DDC, resulting in the formation of Mallory-Denk Bodies (MDBs). In this study, we further discovered that betaine, a methyl donor, fed together with DDC significantly prevents the increased expression of Ufmylation in drug-primed mice fed DDC. Betaine significantly prevented transcript silencing of Ufm1, Uba5 and UfSP1 where MDBs developed and also prevented the increased expression of FAT10 and LMP7 caused by DDC re-fed mice. Similar downregulation of Ufmylation was observed in multiple AH and NASH biopsies which had formed MDBs. The DNA methylation levels of Ufm1, Ufc1 and UfSP1 in the promoter CpG region were significantly increased both in AH and NASH patients compared to normal subjects. DNA (cytosine-5-)-methyltransferase 1 (DNMT1) and DNA (cytosine-5-)-methyltransferase 3 beta (DNMT3B) mRNA levels were markedly upregulated in AH and NASH patients, implying that the maintenance of Ufmylation methylation might be mediated by DNMT1 and DNMT3B together. These data show that MDB formation results from Ufmylation expression epigenetically in AH and NASH patients. Promoter CpG methylation may be a major mechanism silencing Ufmylation expression.

Alcoholic and non-alcoholic steatohepatitis.

This paper is based upon the "Charles Lieber Satellite Symposia" organized by Manuela G. Neuman at the Research Society on Alcoholism (RSA) Annual Meetings, 2013 and 2014. The present review includes pre-clinical, translational and clinical research that characterize alcoholic liver disease (ALD) and non-alcoholic steatohepatitis (NASH). In addition, a literature search in the discussed area was performed. Strong clinical and experimental evidence lead to recognition of the key toxic role of alcohol in the pathogenesis of ALD. The liver biopsy can confirm the etiology of NASH or alcoholic steatohepatitis (ASH) and assess structural alterations of cells, their organelles, as well as inflammatory activity. Three histological stages of ALD are simple steatosis, ASH, and chronic hepatitis with hepatic fibrosis or cirrhosis. These latter stages may also be associated with a number of cellular and histological changes, including the presence of Mallory's hyaline, megamitochondria, or perivenular and perisinusoidal fibrosis. Genetic polymorphisms of ethanol metabolizing enzymes such as cytochrome p450 (CYP) 2E1 activation may change the severity of ASH and NASH. Alcohol mediated hepatocarcinogenesis, immune response to alcohol in ASH, as well as the role of other risk factors such as its co-morbidities with chronic viral hepatitis in the presence or absence of human immunodeficiency virus are discussed. Dysregulation of hepatic methylation, as result of ethanol exposure, in hepatocytes transfected with hepatitis C virus (HCV), illustrates an impaired interferon signaling. The hepatotoxic effects of ethanol undermine the contribution of malnutrition to the liver injury. Dietary interventions such as micro and macronutrients, as well as changes to the microbiota are suggested. The clinical aspects of NASH, as part of metabolic syndrome in the aging population, are offered. The integrative symposia investigate different aspects of alcohol-induced liver damage and possible repair. We aim to (1) determine the immuno-pathology of alcohol-induced liver damage, (2) examine the role of genetics in the development of ASH, (3) propose diagnostic markers of ASH and NASH, (4) examine age differences, (5) develop common research tools to study alcohol-induced effects in clinical and pre-clinical studies, and (6) focus on factors that aggravate severity of organ-damage. The intention of these symposia is to advance the international profile of the biological research on alcoholism. We also wish to further our mission of leading the forum to progress the science and practice of translational research in alcoholism.

The inflammasome in alcoholic hepatitis: Its relationship with Mallory-Denk body formation.

Recent studies indicate that the inflammasome activation plays important roles in the pathogenesis of alcoholic hepatitis (AH). Nod-like receptor protein 3 (NLRP3) is a key component of the macromolecular complex that is so called the inflammasome that triggers caspase 1-dependent maturation of the precursors of IL-1ß and IL-18 cytokines. It is also known that the adaptor proteins including apoptosis-associated speck-like protein containing CARD (ASC) and the mitochondrial antiviral signaling protein (MAVS) are necessary for NLRP3-dependent inflammasome function. Steatohepatitis frequently includes Mallory-Denk body (MDB) formation. In the case of alcoholic steatohepatitis, MDB formation occurs in 80% of biopsies (French 1981; French 1981). While previous studies have focused on in vitro cell lines and mouse models, we are the first group to investigate inflammasome activation in AH liver biopsy specimen and correlate it with MDB formation. Expression of NOD1, NLRP3, ASC, NAIP, MAVS, caspase 1, IL-1ß, IL-18, and other inflammatory components including IL-6, IL-10, TNF-α, IFN-γ, STAT3, and p65 was measured in three to eight formalin-fixed paraffin-embedded AH specimens and control normal liver specimens by immunofluorescence staining and quantified by immunofluorescence intensity. The specimens were double stained with ubiquitin to demonstrate the relationship between inflammasome activation and MDB formation. MAVS, caspase1, IL-18, and TNF-α showed increases in expression in AH compared to the controls (p<0.05), and NAIP expression markedly increased in AH compared to the controls (p<0.01). There was a trend that levels of NLRP3, ASC, caspase1, IL-18, IL-10, and p65 expression correlated with the number of MDBs found in the same field of measurement (correlation coefficients were between 0.62 and 0.93, p<0.05). Our results demonstrate the activation of the inflammasome in AH and suggest that MDB could be an indicator of the extent of inflammasome activation.

Deepwater Horizon crude oil impacts the developing hearts of large predatory pelagic fish.

The Deepwater Horizon disaster released more than 636 million L of crude oil into the northern Gulf of Mexico. The spill oiled upper surface water spawning habitats for many commercially and ecologically important pelagic fish species. Consequently, the developing spawn (embryos and larvae) of tunas, swordfish, and other large predators were potentially exposed to crude oil-derived polycyclic aromatic hydrocarbons (PAHs). Fish embryos are generally very sensitive to PAH-induced cardiotoxicity, and adverse changes in heart physiology and morphology can cause both acute and delayed mortality. Cardiac function is particularly important for fast-swimming pelagic predators with high aerobic demand. Offspring for these species develop rapidly at relatively high temperatures, and their vulnerability to crude oil toxicity is unknown. We assessed the impacts of field-collected Deepwater Horizon (MC252) oil samples on embryos of three pelagic fish: bluefin tuna, yellowfin tuna, and an amberjack. We show that environmentally realistic exposures (1-15 µg/L total PAH) cause specific dose-dependent defects in cardiac function in all three species, with circulatory disruption culminating in pericardial edema and other secondary malformations. Each species displayed an irregular atrial arrhythmia following oil exposure, indicating a highly conserved response to oil toxicity. A considerable portion of Gulf water samples collected during the spill had PAH concentrations exceeding toxicity thresholds observed here, indicating the potential for losses of pelagic fish larvae. Vulnerability assessments in other ocean habitats, including the Arctic, should focus on the developing heart of resident fish species as an exceptionally sensitive and consistent indicator of crude oil impacts.

SAMe treatment prevents the ethanol-induced epigenetic alterations of genes in the Toll-like receptor pathway.

Prior studies showed that Toll-like receptor (TLR) signaling pathway genes were upregulated in the liver of rats fed ethanol, but not in rats fed ethanol plus S-adenosylmethionine (SAMe). These results were obtained using a PCR microplate array analysis for TLRs and associated proteins such as proinflammatory cytokines and chemokine mRNA levels. A large number of genes were upregulated by the ethanol diet, but not the ethanol plus SAMe diet. In the present study, using the same experimental rat livers, DNA methylation analysis was done by using an Epitect Methyl DNA Restriction Kit (Qiagen, 335451) (24 genes). The results of all the genes combined show a highly significant increase in methylation in the ethanol-fed group of rats, but not in the dextrose-fed, SAMe-fed or ethanol plus SAMe-fed groups of rats. There was also an increase in DNA methylation in rats with high blood alcohol levels compared to a rat with a low blood alcohol level. The individual genes that were upregulated as indicated by the increased mRNA measured by qPCR correlated positively with the increased methylation of the DNA of the corresponding genes as follows: Cd14, Hspa1a, Irf1, Irak1, Irak2, Map3k7, Myd88, Pparα, Ripk2, Tollip and Traf6.