Exposure to binge ethanol and fatty acid ethyl esters exacerbates chronic ethanol-induced pancreatic injury in hepatic alcohol dehydrogenase-deficient deer mice.

Alcoholic chronic pancreatitis (ACP) is a fibroinflammatory disease of the pancreas. However, metabolic basis of ACP is not clearly understood. In this study, we evaluated differential pancreatic injury in hepatic alcohol dehydrogenase-deficient (ADH-) deer mice fed chronic ethanol (EtOH), chronic plus binge EtOH, and chronic plus binge EtOH and fatty acid ethyl esters (FAEEs, nonoxidative metabolites of EtOH) to understand the metabolic basis of ACP. Hepatic ADH- and ADH normal (ADH+) deer mice were fed Lieber-DeCarli liquid diet containing 3% (wt/vol) EtOH for 3 mo. One week before the euthanization, chronic EtOH-fed mice were further administered with an oral gavage of binge EtOH with/without FAEEs. Blood alcohol concentration (BAC), pancreatic injury, and inflammatory markers were measured. Pancreatic morphology, ultrastructural changes, and endoplasmic reticulum (ER)/oxidative stress were examined using H&E staining, electron microscopy, immunostaining, and/or Western blot, respectively. Overall, BAC was substantially increased in chronic EtOH-fed groups of ADH- versus ADH+ deer mice. A significant change in pancreatic acinar cell morphology, with mild to moderate fibrosis and ultrastructural changes evident by dilatations and disruption of ER cisternae, ER/oxidative stress along with increased levels of inflammatory markers were observed in the pancreas of chronic EtOH-fed groups of ADH- versus ADH+ deer mice. Furthermore, chronic plus binge EtOH and FAEEs exposure elevated BAC, enhanced ER/oxidative stress, and exacerbated chronic EtOH-induced pancreatic injury in ADH- deer mice suggesting a role of increased body burden of EtOH and its metabolism under reduced hepatic ADH in initiation and progression of ACP.NEW & NOTEWORTHY We established a chronic EtOH feeding model of hepatic alcohol dehydrogenase-deficient (ADH-) deer mice, which mimics several fibroinflammatory features of human alcoholic chronic pancreatitis (ACP). The fibroinflammatory and morphological features exacerbated by chronic plus binge EtOH and FAEEs exposure provide a strong case for metabolic basis of ACP. Most importantly, several pathological and molecular targets identified in this study provide a much broader understanding of the mechanism and avenues to develop therapeutics for ACP.

Polycyclic aromatic hydrocarbons (PAHs) cycling and fates in Galveston Bay, Texas, USA.

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.

Distribution of petrogenic polycyclic aromatic hydrocarbons (PAHs) in seafood following Deepwater Horizon oil spill.

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.

Cytochrome P450 2E1-deficient MRL+/+ mice are less susceptible to trichloroethene-mediated autoimmunity: Involvement of oxidative stress-responsive signaling pathways.

Reactive trichloroethene (TCE) metabolites and oxidative stress are involved in TCE-mediated autoimmunity, as evident from our earlier studies in MRL+/+ mice. However, molecular mechanisms underlying the autoimmunity remain largely unknown. Cytochrome P450 2E1 (CYP2E1), the major enzyme responsible for TCE metabolism, could contribute to TCE-induced toxic response through free radical generation. The current study was, therefore, aimed to further evaluate the significance of TCE metabolism leading to oxidative stress and autoimmune response by using MRL+/+ mice that lack CYP2E1. The Cyp2e1-null MRL+/+ mice were generated by backcrossing Cyp2e1-null mice (B6N; 129S4-Cyp2e1) to MRL +/+ mice. Female MRL+/+ and Cyp2e1-null MRL+/+ mice were given TCE (10 mmol/kg, i.p., every 4th day) for 6 weeks; their respective controls received corn oil only. TCE treatment in MRL+/+ mice induced oxidative stress, evident from significantly increased serum malondiadelhyde (MDA)-protein adducts, their antibodies and reduced liver GSH levels. TCE treatment also modulated Nrf2 pathway with decreased Nrf2 and HO-1, and elevated NF-κB (p65) expression in the liver. TCE exposure also led to increases in serum antinuclear antibodies (ANA) and anti-double stranded DNA antibodies (anti-dsDNA). Although TCE treatment in Cyp2e1-null MRL+/+ mice also led to increases in serum MDA-protein adducts and their antibodies, changes in liver GSH, Nrf2, HO-1 and NF-κB along with increases in serum ANA, anti-dsDNA, the alterations in the oxidative stress and autoimmunity markers in these mice were less pronounced compared to those in MRL+/+ mice. These findings support the contribution of CYP2E1-mediated TCE metabolism in autoimmune response and an important role of Nrf2 pathway in TCE-mediated autoimmunity.

Hepatic alcohol dehydrogenase deficiency induces pancreatic injury in chronic ethanol feeding model of deer mice.

The single most common cause of chronic pancreatitis (CP, a serious inflammatory disease) is chronic alcohol abuse, which impairs hepatic alcohol dehydrogenase (ADH, a major ethanol oxidizing enzyme). Previously, we found ~5 fold greater fatty acid ethyl esters (FAEEs), and injury in the pancreas of hepatic ADH deficient (ADH-) vs. hepatic normal ADH (ADH+) deer mice fed 3.5g% ethanol via liquid diet daily for two months. Therefore, progression of ethanol-induced pancreatic injury was determined in ADH- deer mice fed ethanol for four months to delineate the mechanism and metabolic basis of alcoholic chronic pancreatitis (ACP). In addition to a substantially increased blood alcohol concentration and plasma FAEEs, significant degenerative changes, including atrophy and loss of acinar cells in some areas, ultrastructural changes evident by such features as swelling and disintegration of endoplasmic reticulum (ER) cisternae and ER stress were observed in the pancreas of ethanol-fed ADH- deer mice vs. ADH+ deer mice. These changes are consistent with noted increases in pancreatic injury markers (plasma lipase, pancreatic trypsinogen activation peptide, FAEE synthase and cathepsin B) in ethanol-fed ADH- deer mice. Most importantly, an increased levels of pancreatic glucose regulated protein (GRP) 78 (a prominent ER stress marker) were found to be closely associated with increased phosphorylated eukaryotic initiation factor (eIF) 2α signaling molecule in PKR-like ER kinase branch of unfolded protein response (UPR) as compared to X box binding protein 1S and activating transcription factor (ATF)6 - 50kDa protein of inositol requiring enzyme 1α and ATF6 branches of UPR, respectively, in ethanol-fed ADH- vs. ADH+ deer mice. These results along with findings on plasma FAEEs, and pancreatic histology and injury markers suggest a metabolic basis of ethanol-induced pancreatic injury, and provide new avenues to understand metabolic basis and molecular mechanism of ACP.

Trichloroethene metabolite dichloroacetyl chloride induces apoptosis and compromises phagocytosis in Kupffer Cells: Activation of inflammasome and MAPKs.

Exposure to trichloroethene (TCE), an occupational and ubiquitous environmental contaminant, is associated with the development of several autoimmune diseases, including autoimmune hepatitis (AIH). However, mechanisms contributing to TCE-mediated AIH are not known. Earlier, we have shown that dichloroacetyl chloride (DCAC), one of the reactive metabolites of TCE with strong acylating capability, can elicit an autoimmune response at much lower dose than TCE in female MRL+/+ mice. Furthermore, Kupffer cells (KCs), the liver resident macrophages, are crucial for hepatic homeostasis, but can also participate in the immunopathogenesis of AIH. However, contribution of KCs in TCE-mediated AIH and the underlying mechanisms are not understood. We hypothesized that increased apoptosis and delayed clearance of apoptotic bodies, due to compromised KC function, will result in the breakdown of self-tolerance, autoimmunity, and ultimately AIH. Therefore, using an in vitro model of immortalized mouse KCs, we investigated the contribution of DCAC in TCE-mediated AIH. KCs were treated with different concentrations of DCAC and apoptosis was measured by Annexin V and PI staining. Also, the impact of DCAC on phagocytic potential of KCs was evaluated. Furthermore, markers of inflammasome (NLRP3 and caspase1) were analyzed by real-time PCR and Western blot analysis. DCAC treatment resulted in significantly increased early and late-stage apoptosis, accompanied with inflammasome activation (NLRP3 increases). DCAC treatment resulted in decreased phagocytic function of KCs in a dose-dependent manner, with reduced MFG-E8 levels (phagocytotic function). Furthermore, DCAC exposure led to induction of phos-ERK and phos-AKT signaling. These findings suggest that DCAC induces apoptosis and inflammasome activation, while compromising the phagocytic function of KCs. Our data support that increased apoptosis and impaired KC function by DCAC could be contributory to TCE-mediated AIH.

Alcohol-Induced Hepatic Steatosis: A Comparative Study to Identify Possible Indicator(s) of Alcoholic Fatty Liver Disease.

BACKGROUND: Fatty liver is an early sign of both nonalcoholic and alcoholic fatty liver diseases. Ethanol feeding using a Lieber-DeCarli liquid diet (LD) model which contains 35% fat to rats or mice is a well-established model for alcoholic fatty liver. However, LD diet alone can also induce fatty liver and its differential metabolic profile may be able to differentiate steatosis induced by LD versus LD plus ethanol. PURPOSE: We investigated the lipidomic differences in the livers of Sprague-Dawley (SD) rats fed a pellet diet (PD), LD and liquid ethanol diet (LED) for six weeks. STUDY DESIGN: Male Sprague Dawley rats were fed with nonalcoholic diets PD, LD or LED (ethanol in LD) for six weeks. Lipids were extracted and analyzed by nuclear magnetic resonance (NMR)- based metabolomics. The NMR data obtained was analyzed by multivariate Principal Component Analysis (PCA) and Spotfire DecisionSite 9.0 software to compare PD versus LD and LD versus LED groups. RESULTS: PCA of the NMR spectral data of livers of both comparisons showed a clear separation of PD from LD group and LD from LED group indicating differences in lipid profiles which corresponded with changes in total lipid weights. LD showed increases for cholesterol, esterified cholesterol, cholesterol acetate and triglycerides with decreases for fatty acyl chain, diallylic and allylic protons, while the LED showed increases in esterified cholesterol, cholesterol acetate, fatty acid methyl esters, allylic protons and some triglyceride protons with decreases in free cholesterol and phosphatidylcholine (PC). CONCLUSION: Our data suggest that altered lipid signature or PC levels could be an indicator to differentiate between nonalcoholic versus alcoholic fatty liver.

The Gulf Coast Health Alliance: Health Risks Related to the Macondo Spill (GC-HARMS) Study: Self-Reported Health Effects.

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.

Autoimmune potential of perchloroethylene: Role of lipid-derived aldehydes.

Tetrachloroethene (perchloroethylene, PCE), an ubiquitous environmental contaminant, has been implicated in inducing autoimmunity/autoimmune diseases (ADs), including systemic lupus erythematosus (SLE) and scleroderma in humans. However, experimental evidence suggesting the potential of PCE in mediating autoimmunity is lacking. This study was, therefore, undertaken to explore PCE's potential in inducing/exacerbating an autoimmune response. Six-week old female MRL+/+ mice, in groups of 6 each, were treated with PCE (0.5mg/ml) via drinking water for 12, 18 and 24weeks and markers of autoimmunity and oxidative stress were evaluated. PCE exposure led to significant increases in serum anti-nuclear antibodies (ANA), anti-dsDNA and anti-scleroderma-70 (anti-Scl-70) antibodies at 18weeks and, to a greater extent at 24weeks, suggesting that PCE exposure exacerbated autoimmunity in our animal model. The increases in autoantibodies were associated with time-dependent increases in malondialdehyde (MDA)-protein adducts and their antibodies, as well as significantly decreased levels of antioxidants GSH and SOD. The splenocytes isolated from mice treated with PCE for 18 and 24weeks showed greater Th17 cell proliferation and increased release of IL-17 in culture supernatants following stimulation with MDA-mouse serum albumin adducts, suggesting that MDA-modified proteins may act as an immunologic trigger by activating Th17 cells and contribute to PCE-mediated autoimmunity. Our studies thus provide an experimental evidence that PCE induces/exacerbates an autoimmune response and lipid-derived aldehydes (such as MDA) contribute to this response.

Combining Yeast Display and Competitive FACS to Select Rare Hapten-Specific Clones from Recombinant Antibody Libraries.

The development of antibodies to low molecular weight haptens remains challenging due to both the low immunogenicity of many haptens and the cross-reactivity of the protein carriers used to generate the immune response. Recombinant antibodies and novel display technologies have greatly advanced antibody development; however, new techniques are still required to select rare hapten-specific antibodies from large recombinant libraries. In the present study, we used a combination of phage and yeast display to screen an immune antibody library (size, 4.4 × 10(6)) against hapten markers for petroleum contamination (phenanthrene and methylphenanthrenes). Selection via phage display was used first to enrich the library between 20- and 100-fold for clones that bound to phenanthrene-protein conjugates. The enriched libraries were subsequently transferred to a yeast display system and a newly developed competitive FACS procedure was employed to select rare hapten-specific clones. Competitive FACS increased the frequency of hapten-specific scFvs in our yeast-displayed scFvs from 0.025 to 0.005% in the original library to between 13 and 35% in selected pools. The presence of hapten-specific scFvs was confirmed by competitive ELISA using periplasmic protein. Three distinct antibody clones that recognize phenanthrene and methylphenanthrenes were selected, and their distinctive binding properties were characterized. To our knowledge, these are first antibodies that can distinguish between methylated (petrogenic) versus unmethylated (pyrogenic) phenanthrenes; such antibodies will be useful in detecting the sources of environmental contamination. This selection method could be generally adopted in the selection of other hapten-specific recombinant antibodies.

iNOS null MRL+/+ mice show attenuation of trichloroethene-mediated autoimmunity: contribution of reactive nitrogen species and lipid-derived reactive aldehydes.

Earlier studies from our laboratory in MRL+/+ mice suggest that free radicals, especially overproduction of reactive nitrogen species (RNS) and lipid-derived reactive aldehydes (LDRAs), are associated with trichloroethene (TCE)-mediated autoimmune response. The current study was undertaken to further assess the contribution of RNS and LDRAs in TCE-mediated autoimmunity by using iNOS-null MRL+/+ mice. iNOS-null MRL+/+ mice were obtained by backcrossing iNOS-null mice (B6.129P2-Nos2(tm1Lau)/J) to MRL +/+ mice. Female MRL+/+ and iNOS-null MRL+/+ mice were given TCE (10 mmol/kg, i.p., every 4(th) day) for 6 weeks; their respective controls received corn oil only. TCE exposure led to significantly increased iNOS mRNA in livers, iNOS protein in livers and sera, increased nitrotyrosine (NT) formation in both livers and sera, induction of MDA-/HNE-protein adducts in livers and their respective antibodies in sera along with significant increases in serum antinuclear antibodies (ANA) and anti-dsDNA in MRL+/+ mice. Even though in iNOS-null MRL+/+ mice, the iNOS and NT levels were negligible in both TCE-treated and untreated groups, TCE treatment still led to significant increases in MDA-/HNE-protein adducts and their respective antibodies along with increases in serum ANA and anti-dsDNA compared to controls. Most remarkably, the increases in serum ANA and anti-dsDNA induced by TCE in the iNOS-null MRL+/+ mice were significantly less pronounced compared to that in MRL+/+ mice. Our results provide further evidence that both RNS and LDRAs contribute to TCE-induced autoimmunity in MRL+/+ mice, and iNOS deficiency attenuates this autoimmune response.

Alcoholic Steatosis in Different Strains of Rat: A Comparative Study.

BACKGROUND: Different strains of rats have been used to study alcoholic liver disease (ALD) while the reason for selecting a particular rat strain was not apparent. PURPOSE: The aim of our study was to compare outbred (Wistar) and inbred (Fischer) strains to evaluate pathological, biochemical changes, and gene expression differences associated with ethanol-induced early hepatic steatosis. STUDY DESIGN: Male Wistar and Fischer-344 rats were pair-fed for 6 weeks with or without 5% ethanol in Lieber-DeCarli liquid diet. Livers were analyzed for histological and lipid-related differences. RESULTS: Hepatic midzonal steatosis was mainly found in Wistar rats while Fischer rats showed mostly pericentral steatosis. Increased hepatic steatosis in ethanol-fed Wistar rats is supported by increases in lipids with related genes and transcription factors involved in fatty acid and triglyceride synthesis. CONCLUSION: Our data showed that Fischer rats are relatively less prone to ethanol-mediated steatosis with pericentral lipid deposition pattern in the liver which is similar to humans and show no trace level of lipid accumulation in pair-fed controls as observed in Wistar (outbred) strain. Therefore, Fischer rats are better suited for lipid studies in an early development of ALD.

Nitrosative stress and nitrated proteins in trichloroethene-mediated autoimmunity.

Exposure to trichloroethene (TCE), a ubiquitous environmental contaminant, has been linked to a variety of autoimmune diseases (ADs) including SLE, scleroderma and hepatitis. Mechanisms involved in the pathogenesis of ADs are largely unknown. Earlier studies from our laboratory in MRL+/+ mice suggested the contribution of oxidative/nitrosative stress in TCE-induced autoimmunity, and N-acetylcysteine (NAC) supplementation provided protection by attenuating oxidative stress. This study was undertaken to further evaluate the contribution of nitrosative stress in TCE-mediated autoimmunity and to identify proteins susceptible to nitrosative stress. Groups of female MRL +/+ mice were given TCE, NAC or TCE + NAC for 6 weeks (TCE, 10 mmol/kg, i.p., every 4th day; NAC, ∼ 250 mg/kg/day via drinking water). TCE exposure led to significant increases in serum anti-nuclear and anti-histone antibodies together with significant induction of iNOS and increased formation of nitrotyrosine (NT) in sera and livers. Proteomic analysis identified 14 additional nitrated proteins in the livers of TCE-treated mice. Furthermore, TCE exposure led to decreased GSH levels and increased activation of NF-κB. Remarkably, NAC supplementation not only ameliorated TCE-induced nitrosative stress as evident from decreased iNOS, NT, nitrated proteins, NF-κB p65 activation and increased GSH levels, but also the markers of autoimmunity, as evident from decreased levels of autoantibodies in the sera. These findings provide support to the role of nitrosative stress in TCE-mediated autoimmune response and identify specific nitrated proteins which could have autoimmune potential. Attenuation of TCE-induced autoimmunity in mice by NAC provides an approach for designing therapeutic strategies.

Alcohol oxidizing enzymes and ethanol-induced cytotoxicity in rat pancreatic acinar AR42J cells.

Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disease causing significant morbidity and mortality. Due to lack of a suitable animal model, the underlying mechanism of ACP is poorly understood. Chronic alcohol abuse inhibits alcohol dehydrogenase (ADH) and facilitates nonoxidative metabolism of ethanol to fatty acid ethyl esters (FAEEs) in the pancreas frequently damaged during chronic ethanol abuse. Earlier, we reported a concentration-dependent formation of FAEEs and cytotoxicity in ethanol-treated rat pancreatic tumor (AR42J) cells, which express high FAEE synthase activity as compared to ADH and cytochrome P450 2E1. Therefore, the present study was undertaken to investigate the role of various ethanol oxidizing enzymes in ethanol-induced pancreatic acinar cell injury. Confluent AR42J cells were pre-treated with inhibitors of ADH class I and II [4-methylpyrazole (MP)] or class I, II, and III [1,10-phenanthroline (PT)], cytochrome P450 2E1 (trans-1,2-dichloroethylene) or catalase (sodium azide) followed by incubation with 800 mg% ethanol at 37°C for 6 h. Ethanol metabolism, cell viability, cytotoxicity (apoptosis and necrosis), cell proliferation status, and formation of FAEEs in AR42J cells were measured. The cell viability and cell proliferation rate were significantly reduced in cells pretreated with 1,10-PT + ethanol followed by those with 4-MP + ethanol. In situ formation of FAEEs was twofold greater in cells incubated with 1,10-PT + ethanol and ∼1.5-fold in those treated with 4-MP + ethanol vs. respective controls. However, cells treated with inhibitors of cytochrome P450 2E1 or catalase in combination of ethanol showed no significant changes either for FAEE formation, cell death or proliferation rate. Therefore, an impaired ADH class I-III catalyzed oxidation of ethanol appears to be a key contributing factor in ethanol-induced pancreatic injury via formation of nonoxidative metabolites of ethanol.

N-Acetylcysteine protects against trichloroethene-mediated autoimmunity by attenuating oxidative stress.

Exposure to trichloroethene (TCE), a ubiquitous environmental contaminant, is known to induce autoimmunity both in humans and animal models. However, mechanisms underlying TCE-mediated autoimmunity remain largely unknown. Previous studies from our laboratory in MRL+/+ mice suggest that oxidative stress may contribute to TCE-induced autoimmune response. The current study was undertaken to further assess the role of oxidative stress in TCE-induced autoimmunity by supplementing with an antioxidant N-acetylcysteine (NAC). Groups of female MRL+/+ mice were given TCE, NAC or TCE+NAC for 6 weeks (TCE, 10mmol/kg, i.p., every 4th day; NAC, 250mg/kg/day through drinking water). TCE exposure led to significant increases in serum levels of anti-nuclear, anti-dsDNA and anti-Sm antibodies. TCE exposure also led to significant induction of anti-malondiadelhyde (MDA)- and anti-hydroxynonenal (HNE)-protein adduct antibodies which were associated with increased ANA in the sera along with increased MDA-/HNE-protein adducts in the livers and kidneys, and increases in protein oxidation (carbonylation) in the sera, livers and kidneys, suggesting an overall increase in oxidative stress. Moreover, TCE exposure also resulted in increased release of IL-17 from splenocytes and increases in IL-17 mRNA expression. Remarkably, NAC supplementation attenuated not only the TCE-induced oxidative stress, IL-17 release and mRNA expression, but also the markers of autoimmunity, as evident from decreased levels of ANA, anti-dsDNA and anti-Sm antibodies in the sera. These results provide further support to a role of oxidative stress in TCE-induced autoimmune response. Attenuation of TCE-induced autoimmunity in mice by NAC provides an approach for preventive and/or therapeutic strategies.

Liver proteomics in progressive alcoholic steatosis.

Fatty liver is an early stage of alcoholic and nonalcoholic liver disease (ALD and NALD) that progresses to steatohepatitis and other irreversible conditions. In this study, we identified proteins that were differentially expressed in the livers of rats fed 5% ethanol in a Lieber-DeCarli diet daily for 1 and 3 months by discovery proteomics (two-dimensional gel electrophoresis and mass spectrometry) and non-parametric modeling (Multivariate Adaptive Regression Splines). Hepatic fatty infiltration was significantly higher in ethanol-fed animals as compared to controls, and more pronounced at 3 months of ethanol feeding. Discovery proteomics identified changes in the expression of proteins involved in alcohol, lipid, and amino acid metabolism after ethanol feeding. At 1 and 3 months, 12 and 15 different proteins were differentially expressed. Of the identified proteins, down regulation of alcohol dehydrogenase (-1.6) at 1 month and up regulation of aldehyde dehydrogenase (2.1) at 3 months could be a protective/adaptive mechanism against ethanol toxicity. In addition, betaine-homocysteine S-methyltransferase 2 a protein responsible for methionine metabolism and previously implicated in fatty liver development was significantly up regulated (1.4) at ethanol-induced fatty liver stage (1 month) while peroxiredoxin-1 was down regulated (-1.5) at late fatty liver stage (3 months). Nonparametric analysis of the protein spots yielded fewer proteins and narrowed the list of possible markers and identified d-dopachrome tautomerase (-1.7, at 3 months) as a possible marker for ethanol-induced early steatohepatitis. The observed differential regulation of proteins have potential to serve as biomarker signature for the detection of steatosis and its progression to steatohepatitis once validated in plasma/serum.

Hepatic lipid profiling of deer mice fed ethanol using ¹H and ³¹P NMR spectroscopy: a dose-dependent subchronic study.

Chronic alcohol abuse is a 2nd major cause of liver disease resulting in significant morbidity and mortality. Alcoholic liver disease (ALD) is characterized by a wide spectrum of pathologies starting from fat accumulation (steatosis) in early reversible stage to inflammation with or without fibrosis and cirrhosis in later irreversible stages. Previously, we reported significant steatosis in the livers of hepatic alcohol dehydrogenase (ADH)-deficient (ADH⁻) vs. hepatic ADH-normal (ADH⁺) deer mice fed 4% ethanol daily for 2 months [Bhopale et al., 2006, Alcohol 39, 179-188]. However, ADH⁻ deer mice fed 4% ethanol also showed a significant mortality. Therefore, a dose-dependent study was conducted to understand the mechanism and identify lipid(s) involved in the development of ethanol-induced fatty liver. ADH⁻ and ADH⁺ deer mice fed 1, 2 or 3.5% ethanol daily for 2 months and fatty infiltration in the livers were evaluated by histology and by measuring dry weights of extracted lipids. Lipid metabolomic changes in extracted lipids were determined by proton (¹H) and ³¹phosphorus (³¹P) nuclear magnetic resonance (NMR) spectroscopy. The NMR data was analyzed by hierarchical clustering (HC) and principle component analysis (PCA) for pattern recognition. Extensive vacuolization by histology and significantly increased dry weights of total lipids found only in the livers of ADH⁻ deer mice fed 3.5% ethanol vs. pair-fed controls suggest a dose-dependent formation of fatty liver in ADH⁻ deer mouse model. Analysis of NMR data of ADH⁻ deer mice fed 3.5% ethanol vs. pair-fed controls shows increases for total cholesterol, esterified cholesterol, fatty acid methyl esters (FAMEs), triacylglycerides and unsaturation, and decreases for free cholesterol, phospholipids and allylic and diallylic protons. Certain classes of neutral lipids (cholesterol esters, fatty acyl chain (-COCH2-) and FAMEs) were also mildly increased in ADH⁻ deer mice fed 1 or 2% ethanol. Only small increases were observed for allylic and diallylic protons, FAMEs and unsaturations in ADH⁺ deer mice fed 3.5% ethanol vs. pair-fed controls. PCA of NMR data showed increased clustering by gradual separation of ethanol-fed ADH⁻ deer mice groups from their respective pair-fed control groups and corresponding ethanol-fed ADH⁺ deer mice groups. Our data indicate that dose of ethanol and hepatic ADH deficiency are two key factors involved in initiation and progression of alcoholic fatty liver disease. Further studies on characterization of individual lipid entities and associated metabolic pathways altered in our deer mouse model after different durations of ethanol feeding could be important to delineate mechanism(s) and identify potential biomarker candidate(s) of early stage ALD.

Remineralization Effect of Topical NovaMin Versus Sodium Fluoride (1.1%) on Caries-Like Lesions in Permanent Teeth.

OBJECTIVE: NovaMin, a synthetic mineral composed of calcium, sodium, phosphorous and silica releases deposits of crystalline hydroxyl-carbonate apatite (HCA) structurally similar to tooth mineral composition. The aim of this investigation was to compare the potential remineralization effect of topical NovaMin and Sodium Fluoride gel on caries like lesions in permanent teeth. MATERIALS AND METHODS: A total of 60 sound human freshly extracted teeth were subjected to a pH-cycling protocol. Specimens were randomly assigned to one of the two treatment groups with NovaMin contained dentifrice applied to group 1, while group 2 received a dentifrice containing 1.1% neutral Sodium Fluoride. Pastes were applied five times after the samples received a demineralization from an earlier cariogenic challenge. Specimens were then evaluated by a Surface Micro Hardness test (SMH, 25G, 5s). Post-treatment SMH measurements were conducted and Mann Whitney test was employed for statistical analysis. RESULTS: Mean post lesion SMH values were 221.99±26.27 and 224.50±28.64 for the first and second groups, respectively. Post treatment SMH values were 232.52±24.34 for NovaMin and 232.03 ±24.46 for the fluoride group. Two way ANOVA test showed a highly significant difference between the two different treatment protocols (p<0.001). CONCLUSION: NovaMin dentifrice appears to have a greater effect on remineralization of carious-like lesions when compared to that of fluoride containing dentifrice in permanent teeth.

Protein adducts of malondialdehyde and 4-hydroxynonenal contribute to trichloroethene-mediated autoimmunity via activating Th17 cells: dose- and time-response studies in female MRL+/+ mice.

Trichloroethene (TCE), a common occupational and environmental toxicant, is known to induce autoimmunity. Previous studies in our laboratory showed increased oxidative stress in TCE-mediated autoimmunity. To further establish the role of oxidative stress and to investigate the mechanisms of TCE-mediated autoimmunity, dose- and time-response studies were conducted in MRL+/+ mice by treating them with TCE via drinking water at doses of 0.5, 1.0 or 2.0mg/ml for 12, 24 or 36 weeks. TCE exposure led to dose-related increases in malondialdehyde (MDA)-/hydroxynonenal (HNE)-protein adducts and their corresponding antibodies in the sera and decreases in GSH and GSH/GSSG ratio in the kidneys at 24 and 36 weeks, with greater changes at 36 weeks. The increases in these protein adducts and decreases in GSH/GSSG ratio were associated with significant elevation in serum anti-nuclear- and anti-ssDNA-antibodies, suggesting an association between TCE-induced oxidative stress and autoimmune response. Interestingly, splenocytes from mice treated with TCE for 24 weeks secreted significantly higher levels of IL-17 and IL-21 than did splenocytes from controls after stimulation with MDA-mouse serum albumin (MSA) or HNE-MSA adducts. The increased release of these cytokines showed a dose-related response and was more pronounced in mice treated with TCE for 36 weeks. These studies provide evidence that MDA- and or HNE-protein adducts contribute to TCE-mediated autoimmunity, which may be via activation of Th17 cells.

1H Nuclear Magnetic Resonance (NMR) Metabolomic Study of Chronic Organophosphate Exposure in Rats.

1H NMR spectroscopy and chemometric analysis were used to characterize rat urine obtained after chronic exposure to either tributyl phosphate (TBP) or triphenyl phosphate (TPP). In this study, the daily dose exposure was 1.5 mg/kg body weight for TBP, or 2.0 mg/kg body weight for TPP, administered over a 15-week period. Orthogonal signal correction (OSC) -filtered partial least square discriminant analysis (OSC-PLSDA) was used to predict and classify exposure to these organophosphates. During the development of the model, the classification error was evaluated as a function of the number of latent variables. NMR spectral regions and corresponding metabolites important for determination of exposure type were identified using variable importance in projection (VIP) coefficients obtained from the OSC-PLSDA analysis. As expected, the model for classification of chronic (1.5-2.0 mg/kg body weight daily) TBP or TPP exposure was not as strong as the previously reported model developed for identifying acute (15-20 mg/kg body weight) exposure. The set of majorly impacted metabolites identified for chronic TBP or TPP exposure was slightly different than those metabolites previously identified for acute exposure. These metabolites were then mapped to different metabolite pathways and ranked, allowing the metabolic response to chronic organophosphate exposure to be addressed.