Biomedical subjects

Terrence M Donohue

Publications and source records attributed to Terrence M Donohue.

14 recordsLinked to original sources

PubMed2007

Ethanol-induced oxidative stress suppresses generation of peptides for antigen presentation by hepatoma cells.

UNLABELLED: Processing of peptides for antigen presentation is catalyzed by antigen-trimming enzymes, including the proteasome and leucine aminopeptidase. Oxidative stress suppresses proteasome function. We hypothesized that in liver cells, processing of antigenic peptides is altered by ethanol metabolism. To address this issue, soluble extracts of ethanol-metabolizing VL-17A cells treated with 100 mM ethanol or left untreated were incubated with C-extended or N-extended 18-27 HBV core peptides. Peptide cleavage was measured by recovery after HPLC. Ethanol exposure to VL-17A cells increased CYP2E1 and decreased proteasome peptidase activities. The latter effect was prevented by treatment of cells with inhibitors, 4-methylpyrazole and diallyl sulfide. Ethanol treatment of VL-17A cells also reduced the activity of leucine aminopeptidase (LAP). Consequently, cleavage of both C-extended and N-extended peptides by cytosolic extracts was suppressed by pretreatment of cells with ethanol. Treatment of cells with interferon gamma, which enhances proteasome activity, did not reverse the effects of ethanol. Ethanol exerted similar effects on WIFB cells, indicating that its effects are not unique to one cell type. CONCLUSION: Ethanol metabolism suppresses activities of antigen-trimming enzymes, thereby decreasing the cleavage of C-extended and N-extended peptides. This defect may potentially result in decreased MHC class I-restricted antigen presentation on virally infected liver cells.

Antigen Presentation↗

PubMed2005

Recombinant Hep G2 cells that express alcohol dehydrogenase and cytochrome P450 2E1 as a model of ethanol-elicited cytotoxicity.

HepG2 cells were transfected with recombinant plasmids, one carrying the murine alcohol dehydrogenase (ADH) gene and the other containing the gene encoding human cytochrome P450 2E1 (CYP2E1). One of recombinant clones called VL-17A exhibited ADH and CYP2E1 specific activities comparable to those in isolated rat hepatocytes. VL-17A cells oxidized ethanol and generated acetaldehyde, the levels of which depended upon the initial ethanol concentration. Compared with unexposed VL-17A cells, ethanol exposure increased the cellular redox (lactate:pyruvate ratio) and caused cell toxicity, indicated by increased leakage of lactate dehydrogenase into the medium,. Exposure of VL-17A cells to 100mM ethanol significantly elevated caspase 3 activity, an indicator of apoptosis, but this ethanol concentration did not affect caspase 3 activity in parental HepG2 cells. Because ethanol consumption causes a decline in hepatic protein catabolism, we examined the influence of ethanol exposure on proteasome activity in HepG2, VL-17A, E-47 (CYP2E1(+)) and VA-13 (ADH(+)) cells. Exposure to 100mM ethanol caused a 25% decline in the chymotrypsin-like activity of the proteasome in VL-17A cells, but the enzyme was unaffected in the other cell types. This inhibitory effect on the proteasome was blocked when ethanol metabolism was blocked by 4-methyl pyrazole. We conclude that recombinant VL-17A cells, which express both ADH and CYP2E1 exhibit hepatocyte-like characteristics in response to ethanol. Furthermore, the metabolism of ethanol by these cells via ADH and CYP2E1 is sufficient to bring about an inhibition of proteasome activity that may lead to apoptotic cell death.

Alcohol Dehydrogenase↗

PubMed2005

Ethanol metabolism alters interferon gamma signaling in recombinant HepG2 cells.

We previously showed that IFNgamma signal transduction was suppressed by ethanol in recombinant HepG2 cells (VL-17A cells), which express alcohol dehydrogenase (ADH) and CYP2E1. We examined the mechanisms by which STAT1 phosphorylation is blocked by ethanol treatment in VL-17A cells. Cells were exposed to 0 or 100 mmol/L ethanol for 72 hours. STAT1 phosphorylation was determined by Western blot after 1 hour IFNgamma exposure. Reduction of STAT1 phosphorylation by ethanol was prevented in the presence of 4MP, DAS, or uric acid, indicating that the oxidative products from ethanol metabolism were partly responsible for suppression of STAT1 phosphorylation. Ethanol exposure decreased STAT1 tyrosine phosphorylation, whereas serine phosphorylation on the protein was unchanged. These effects of ethanol were mimicked by the peroxynitrite (PN) donor, SIN-1, which also blocked tyrosine, but not serine phosphorylation, on STAT1. When cells expressing either ADH (VA-13 cells) or CYP2E1 (E-47 cells) were exposed to ethanol, both ADH- and CYP2E1-generated products reduced STAT1 phosphorylation. In addition, SOCS1, a negative regulator of IFNgamma signaling and which is degraded by the proteasome, was stabilized by ethanol treatment, presumably because of inhibited proteasome activity. Furthermore, SIN-1 treatment elevated SOCS1 levels in VL-17A cells, indicating that PN has a role in SOCS1 elevation. In conclusion, under conditions of ethanol-elicited oxidative stress, PN prevents STAT1 phosphorylation by stabilization of SOCS1, and possibly by nitration of tyrosine residues in STAT1 protein.

Blotting, Western↗

PubMed2005

Inhibition of bone repair in a rat model for chronic and excessive alcohol consumption.

Alcohol abuse is associated with increases in both the incidence of fractures and complications in fracture healing. The purpose of this study was to determine the dose-dependent effects of ethanol on bone repair in a rat model. Three-month-old male Wistar rats were continuously fed liquid diets containing ethanol as either 36% or 26% of total calories or control diets for 6 weeks. Then, a bone repair model was created in all rats. Bone healing and liver metabolism were evaluated 7 weeks after bone injury. For each dose, there were three ethanol-feeding groups receiving (1) ethanol for 13 weeks, (2) control diet for 13 weeks (pair-fed), and (3) ethanol before bone injury and control diet (pair-fed) after injury. Another group was fed ethanol (36%) before injury and given control diet ad libitum after injury. There were also two nutritional controls consuming control diet and standard rat chow ad libitum for 13 weeks. Abnormal liver metabolism was evident at the higher ethanol dose - increases in cytochrome P4502E1 specific activity (5-fold; P < .01), triglyceride content (4-fold; P < .02), and liver weight (25%; P = .05) - compared with pair-fed controls. The higher dose of ethanol resulted in deficient bone repair when compared with rats receiving ethanol-free control diet by pair-feeding: 26% less (P = .02) rigidity of the repaired bone, 41% less (P = .02) intrinsic stiffness, 24% less intrinsic strength (P = .05), and 14% less (P = .001) ash density of the repair tissue. The reduced food consumption of ethanol-fed rats compared with that in the nutritional controls did not contribute to this deficiency. Furthermore, removal of ethanol (as 36% of calories) from the diet after bone injury completely restored normal bone healing and nearly normalized the liver metabolism. The lower ethanol dose (26% of calories) had a minimal effect on liver metabolism and bone repair. We conclude that ethanol (as 36% of calories) in the rat diet, especially during the postinjury period, was solely responsible for the observed inhibition of bone repair.

Alcoholism↗

PubMed2004

Peroxynitrite alters the catalytic activity of rodent liver proteasome in vitro and in vivo.

The proteasome is an important multicatalytic enzyme complex that degrades misfolded and oxidized proteins, signal transduction factors, and antigenic peptides for presentation. We investigated the in vitro effects of peroxynitrite (PN) on the peptidase activity of both crude 20S and 26S and purified 20S proteasome preparations from rat liver as well as proteasome activity in Hep G2 cells and in mouse liver. Crude and purified proteasome preparations were exposed to PN or to the PN donor, 3-morpholinosydnonimine hydrochloride (SIN-1), and then assayed for chymotrypsin-like activity. For in vivo experiments, mice were treated with molsidomine, which is metabolized to SIN-1 in liver. PN and SIN-1 dose-dependently modulated the chymotrypsin-like activity of the 20S proteasome: lower concentrations enhanced proteasome activity, and higher concentrations caused its decline. The NO donor S-nitroso-N-acetylpenicillamine (SNAP), at all concentrations, suppressed 20S proteasome activity. We observed similar results when liver soluble fractions (S-100) were treated with PN, SIN-1, or SNAP, except that enzyme activity in S-100 fractions was less sensitive than the purified enzymes to these agents. Treatment of Hep G2 cells with 0.01 or 0.1 mmol/L SIN-1 stimulated in situ proteasome activity in these cells, while 1 mmol/L SIN-1 suppressed it. SNAP treatment did not affect proteasome activity in Hep G2 cells. Mice treated with molsidomine had enhanced liver proteasome activity 6 hours after treatment, but after 24 hours enzyme activity declined below control levels. In conclusion, PN dose-dependently modulated proteasome activity, regulating protein degradation by the proteasome in liver cells.

Animals↗

PubMed2004

Alcohol and HIV decrease proteasome and immunoproteasome function in macrophages: implications for impaired immune function during disease.

Proteasomes (proteinase complexes, PR) and immunoproteasomes (IPR) degrade damaged proteins and affect protein processing required for antigen presentation by mononuclear phagocytes. These critical immune processes are attenuated during progressive HIV-1 infection and are affected by alcohol abuse. To investigate the mechanisms underlying these functional changes, we measured PR and CYP2E1 activities [an ethanol (EtOH) metabolizing enzyme] and reactive oxygen species (ROS) in human monocyte-derived macrophages (MDM) following HIV-1 infection and EtOH treatment. We observed progressive declines of PR activity and PR/IPR contents in HIV-1-infected MDM. PR activity and IPR expression increased after IFN-gamma stimulation but reduced after HIV-1 infection. EtOH inhibited both IFN-gamma-induced PR and IPR. Paradoxically, EtOH attenuated PR catalytic activity in infected MDM and suppressed viral replication. Elevated ROS followed EtOH exposure and paralleled decreased PR activity. The latter was restored by anti-oxidant. The data support the notion that HIV-1 infection and EtOH may work in concert to affect immune function including antigen presentation and thereby affect disease progression.

Alcoholism↗

PubMed2004

Decreased proteasome activity is associated with increased severity of liver pathology and oxidative stress in experimental alcoholic liver disease.

BACKGROUND: Because of its role in degrading the bulk of intracellular proteins and eliminating damaged proteins, the proteasome is important in maintaining cell viability. Previously, we showed a 35-40% decrease in proteasome peptidase activity when ethanol was administered to rats by intragastric infusion. We hypothesized that this reduction was caused by ethanol-elicited oxidative stress, the degree of which varies depending on the method of ethanol administration. This study examined the relationship of proteasome activity and content with ethanol-induced oxidative stress and the degree of liver injury. METHODS: Rats were given ethanol or isocaloric dextrose-containing liquid diets by intragastric infusion for 1 month. The diets contained medium-chain triglycerides (MCT), palm oil (PO), corn oil (CO), or fish oil (FO) as the principal source of fat. RESULTS: Rats given ethanol and MCT exhibited no significant liver pathology, whereas cumulative pathology scores in ethanol-fed rats given PO, CO, or FO were 2.5, 5.4 and 7.0, respectively, indicating that ethanol and FO caused the greatest liver damage. The severity of liver pathology in the last three groups of animals correlated with levels of lipid peroxides and serum 8-isoprostanes. Alpha smooth muscle actin, an indicator of stellate cell activation, was increased relative to controls in the livers of all ethanol-fed rats except FO-fed animals, in which both control and ethanol-fed rats had similar levels of this protein. In livers of CO and FO ethanol-fed rats, proteasome chymotrypsin-like activity was decreased by 55-60%, but there was no quantitative alteration in 20S proteasome subunit content. In contrast, ethanol affected neither proteasome activity nor its content in MCT- and PO-treated animals. CONCLUSIONS: Our findings indicate that the severity of liver injury and ethanol-induced oxidative stress is associated with a reduction in proteasome catalysis.

Animals↗

PubMed2003

Interferon gamma enhances proteasome activity in recombinant Hep G2 cells that express cytochrome P4502E1: modulation by ethanol.

We tested the influence of IFNgamma on proteasome activity in parental Hep G2 cells that do not metabolize ethanol, as well as in recombinant Hep G2-derived cells that express either or both alcohol dehydrogenase (ADH) and cytochrome P4502E1 (CYP2E1). IFNgamma treatment increased proteasome activity in VL-17A (ADH(+), CYP2E1(+)) and E-47 (CYP2E1(+)) cells, but not in Hep G2, VI-R2 (parental cells with empty vectors) or in VA-13 (ADH(+)) cells. Proteasome activation by IFNgamma correlated positively with the level of CYP2E1 activity. Treatment of VL-17A cells with agents that inhibit CYP2E1 or the inducible nitric oxide synthase (iNOS) or that prevent the formation of peroxynitrite also blocked proteasome activation by IFNgamma, indicating that the proteasome may be directly activated by products of CYP2E1 and iNOS catalysis. While IFNgamma treatment increased proteasome activity, it also decreased CYP2E1 activity. Both effects were mediated via the Janus kinase-signal transducer and activator of transcription 1 (JAK-STAT1) pathway, as both were blocked by the JAK2 inhibitor, tyrphostin AG 490. Ethanol treatment of VL-17A cells also caused a similar blockage of these same IFNgamma-mediated effects, by inhibiting STAT1 phosphorylation. This inhibition was largely due to ethanol metabolism, as 4-methylpyrazole, an ethanol metabolism inhibitor, restored IFNgamma-mediated STAT1 phosphorylation in ethanol-treated cells. Our results lead us to propose that IFNgamma initiates signal transduction, which alters the activities of CYP2E1 and iNOS, thereby producing reactive oxygen species. One of these oxidants, possibly peroxynitrite, may be directly involved in proteasome activation. Ethanol metabolism by VL-17A cells suppresses IFNgamma-mediated induction of proteasome activity, in part, by preventing STAT1 phosphorylation.

Cysteine Endopeptidases↗

PubMed2003

Liver asialoglycoprotein receptor levels correlate with severity of alcoholic liver damage in rats.

It has been demonstrated that the oral administration of ethanol (Lieber-DeCarli liquid diet) to rats results in a decreased expression and content of the asialoglycoprotein receptor (ASGP-R) in the resultant fatty liver. In the present study, we wanted to determine whether the extent of impaired receptor content was correlated with the severity of liver pathology by using the intragastric feeding model. When ASGP-R protein and mRNA levels were measured in animals infused with ethanol or dextrose in the presence of fish oil (FO) or medium-chain triglyceride as the source of fat, more significant impairments to the ASGP-R were observed in the FO-ethanol group compared with the medium-chain triglyceride-ethanol group. Furthermore, only the FO-ethanol group showed pathological liver changes. These results demonstrate that a correlation exists between the progression of alcohol-associated liver injury, as defined by the severity of liver pathology, and an ethanol-induced decline in ASGP-R content.

Animal Feed↗

PubMed2003

Ethanol consumption decreases the synthesis of the mannose 6-phosphate/insulin-like growth factor II receptor but does not decrease its messenger RNA.

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-IIR) is a protein that facilitates the transport of acid hydrolases into the lysosome. We have shown that chronic ethanol consumption lowers the M6P/IGF-IIR content in rat hepatocytes. Here, we determined the steady-state level of mRNA encoding M6P/IGF-IIR, as well as the rate of receptor synthesis, to ascertain whether the ethanol-elicited reduction in receptor protein content is related to changes in either or both of these parameters. Rats were pair-fed the normal carbohydrate (NC) or low carbohydrate high-fat (LC) liquid diets containing either ethanol or isocaloric maltose-dextrin for 7-8 weeks. RNA was isolated from hepatocytes and from whole livers of these animals and subjected to reverse transcription-polymerase chain reaction (RT-PCR) to determine the mRNA levels encoding M6P/IGF-IIR. Hepatocytes isolated from these animals were also radiolabeled with Pro-mix L-[35S] in vitro cell labeling mix to measure incorporation into total cellular protein and the immunoprecipitated M6P/IGF-IIR protein. The steady-state levels of M6P/IGF-IIR mRNA in both hepatocytes and whole livers from ethanol-fed rats were the same as those from their respective controls regardless of whether they were fed the NC or the LC diets. Hepatocytes from ethanol-fed rats showed a 36% lower rate of total protein synthesis and an even greater reduction (70%) in receptor synthesis. When the relative rate of receptor synthesis was calculated, hepatocytes from ethanol-fed rats had a 53% lower relative rate of receptor synthesis compared with controls. Autoradiographic analysis of the immunoprecipitated receptor protein from ethanol-fed rats also indicated a 79% decline in the total M6P/IGF-IIR protein synthetic rate compared with pair-fed controls. We conclude that the ethanol-elicited reduction of M6P/IGF-IIR content was, in part, related to a concomitant reduction of receptor protein synthesis but not to a decline in its mRNA level. Thus, the ethanol-elicited decline in receptor protein synthesis may be due to defective M6P/IGF-IIR mRNA translation.

Alcohol Drinking↗

PubMed2003

Intracellular proteolytic systems in alcohol-induced tissue injury.

The body constantly produces proteins and degrades proteins that are no longer needed or are defective. The process of protein breakdown, called proteolysis, is essential to cell survival. Numerous proteolytic systems exist in mammalian cells, the most important of which are the lysosomes, the ubiquitin-proteasome pathway, and enzymes called calpains. Lysosomes are small cell components that contain specific enzymes (i.e., proteases) which break down proteins. Alcohol interferes with the formation and activity of lysosomes and thus may contribute to protein accumulation in the liver, which can have harmful effects on that organ. In the ubiquitin-proteasome pathway, proteins that are to be degraded are first marked by the addition of ubiquitin molecules and then broken down by large protein complexes called proteasomes. Alcohol impairs this proteolytic system through several mechanisms, possibly leading to inflammation and even cell death. Calpains are proteases that are involved in several physiological processes, including the breakdown of proteins that give cells their shape and stability. In contrast to the lysosomal and ubiquitin-proteasome systems, calpains in brain cells are activated by alcohol, to potentially detrimental effect.

Alcohol-Induced Disorders↗

PubMed2002

Chronic ethanol administration decreases the ligand binding properties and the cellular content of the mannose 6-phosphate/insulin-like growth factor II receptor in rat hepatocytes.

We have shown previously that chronic ethanol administration impairs the maturation of lysosomal enzymes in rat hepatocytes. The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-IIR) is a protein that facilitates the transport of lysosomal enzymes into the lysosome. Therefore, we examined whether ethanol consumption altered the ligand binding properties and the cellular content of M6P/IGF-IIR. Rats were pair-fed liquid diets containing either ethanol (36% of calories) or isocaloric maltose-dextrin for either 1 week or 5-7 weeks. Hepatocytes prepared from these animals were examined for receptor-ligand binding and receptor content. One week of ethanol feeding had no significant effect on ligand [radioiodinated pentamannose phosphate conjugated to bovine serum albumin ((125)I-PMP-BSA)] binding to hepatocytes, but cells from rats fed ethanol for 5-7 weeks bound less (125)I-PMP-BSA than pair-fed controls. Scatchard plot analysis revealed that the number of (125)I-PMP-BSA binding sites in hepatocytes from ethanol-fed rats was 49% lower than that of controls. (125)I-PMP-BSA binding by perivenular (PV) and periportal (PP) hepatocytes from ethanol-fed rats was, respectively, 40 and 48% lower than their controls, but there was no significant difference between these two types of hepatocytes. Ligand blot analysis using (125)I-insulin-like growth factor II ((125)I-IGF-II) also showed that the receptor in lysates of hepatocytes from ethanol-fed rats bound 26-27% less ligand than controls. Similarly, immunoblot analysis of cell lysates from ethanol-fed rats revealed 62% lower levels of immunoreactive M6P/IGF-IIR than controls. Feeding rats a low carbohydrate-ethanol diet did not exacerbate the reduction in M6P/IGF-IIR-ligand binding nor did it reduce the levels of immunoreactive receptor. Our findings indicate that chronic ethanol consumption lowers M6P/IGF-IIR activity and content in hepatocytes. This reduction may account, in part, for the impaired processing and delivery of acid hydrolases to lysosomes previously observed in ethanol-fed rats.

Animals↗

PubMed2002

The ubiquitin-proteasome system and its role in ethanol-induced disorders.

Some of the most fundamental yet important cellular activities such as cell division and gene expression are controlled by short-lived regulatory proteins. The levels of these proteins are controlled by their rates of degradation. Similarly, protein catabolism plays a crucial role in prolonging cellular life by destroying damaged proteins that are potentially cytotoxic. A major player in these catabolic reactions is the ubiquitin-proteasome system, a novel proteolytic system that has become the primary proteolytic pathway in eukaryotic cells. Ubiquitin-mediated proteolysis is now regarded as the major pathway by which most intracellular proteins are destroyed. Equally important, from a toxicological standpoint, is that the ubiquitin-proteasome system is also widely considered to be a cellular defense mechanism, since it is involved in the removal of damaged proteins generated by adduct formation and oxidative stress. This review describes the history and the components of the ubiquitin-proteasome system, its regulation and its role in pathological states, with the major emphasis on ethanol-induced organ injury. The available literature cited here deals mainly with the effects of ethanol consumption on the ubiquitin-proteasome pathway in the liver. However, since this proteolytic system is an essential pathway in all cells it is an attractive experimental model and therapeutic target in extrahepatic organs such as the brain and heart that are also affected by excessive alcohol consumption.

Alcohol Amnestic Disorder↗

PubMedDate not supplied

Chronic ethanol consumption results in deficient bone repair in rats.

There is evidence that ethanol inhibits osteoblast function and that chronic ethanol consumption induces systemic bone loss and increases the risk of fracture in humans. The purpose of the present study was to determine whether chronic ethanol consumption also compromises the healing of injured bone. Male Sprague-Dawley rats, 8-10 weeks old, were placed into four feeding groups: group A received ethanol (36% of calories) as part of a liquid diet; group B was pair-fed to group A and received an isocaloric control diet containing maltodextrin; group C was fed the AIN-93M standard semi-purified liquid diet ad libitum; group D was fed the same ethanol diet as group A before bone injury, but after surgery (see below) these rats were given isocaloric control diet ad libitum. After 6 weeks on their respective diets, a bone repair model was surgically created at the midshaft in both fibulae of each rat. Seven weeks after injury the animals were euthanized and bone healing was evaluated by determining rigidity of the fibula by three-point bending, flexural modulus of the repair tissue and mineral content of the repair tissue. Rigidity of fibula in ethanol-fed rats and their pair-fed controls (groups A and B) were respectively 48 and 47% lower than in group C. Flexural modulus of the repair tissue in ethanol-fed rats had a 55% (P = 0.046) deficiency compared with their pair-fed controls. The mineral contents in groups A and B were respectively 16 and 13% lower than in group C. There were no significant differences in the results between groups C and D. Thus, the outcome of bone repair in ethanol-fed rats was deficient compared with rats receiving a standard maintenance diet. The repair tissue in ethanol-fed rats was mechanically inferior to that in pair-fed controls. This deficiency could not be attributed to the reduced food consumption of these animals. On the other hand, the restoration of normal bone healing in group D cannot be attributed solely to the cessation of ethanol feeding after bone injury because of the increased food consumption during this period.

Alcoholism↗