Hemolytic iron regulation in traumatic brain injury and alcohol use.

Hemorrhage is a major component of traumatic brain injury (TBI). Red blood cells, accumulated at the hemorrhagic site, undergo hemolysis upon energy depletion and release free iron into the central nervous system. This iron must be managed to prevent iron neurotoxicity and ferroptosis. As prior alcohol consumption is often associated with TBI, we examined iron regulation in a rat model of chronic alcohol feeding subjected to fluid percussion-induced TBI. We found that alcohol consumption prior to TBI altered the expression profiles of the lipocalin 2/heme oxygenase 1/ferritin iron management system. Notably, unlike TBI alone, TBI following chronic alcohol consumption sustained the expression of all three regulatory proteins for 1, 3, and 7 days post-injury. In addition, alcohol significantly affected TBI-induced expression of ferritin light chain at 3 days post-injury. We also found that alcohol exacerbated TBI-induced activation of microglia at 7 days post-injury. Finally, we propose that microglia may also play a role in iron management through red blood cell clearance.

Synergistic effects of alcohol and HIV TAT protein on macrophage migration and neurotoxicity.

The trans-activator of transcription (TAT) is a human immunodeficiency virus (HIV-1) regulatory protein that is actively sloughed by infected cells. Once released, TAT can injure bystander cells and bring about their dysfunction. In the presence of ethanol, TAT-induced toxicity potentiates and, in so doing, exacerbates inflammation. One key aspect of neuroinflammation involves the infiltration of peripheral macrophage to the central nervous system. Here, we use an interactive neuroimmune cell coculture of brain endothelial, astrocyte, neuron, and macrophage cells to model the blood-brain barrier and evaluate macrophage migration upon challenge with ethanol and TAT concentrations. We have limited this study to examine TAT concentrations found in people living with HIV-1 with (5 ng/mL) or without (25 ng/mL) viral suppression and ethanol doses below the legal driving limit (10 mM). In so doing, we study the effects of casual drinking on people living with HIV-1 but experiencing the best possible clinical outcome. We found that TAT alone increases macrophage migration between 0.5 and 4 h. while ethanol alone increases migration in a delayed manner (occurring at 48 h.). Ethanol-induced NO production by endothelial cells and TAT's chemoattractant properties may explain this dichotomy in migration pattern. Combined low dose ethanol significantly increased migration under both 5 ng/mL and 25 ng/mL TAT injuries across all timepoints. Our findings suggest that co-presence of ethanol and TAT may be the combination of an initial TAT effect followed by subsequent ethanol treatment. We also examined the structural and behavioral changes of neurons treated with TAT and ethanol to understand their contribution to neurotoxicity. The lowest concentration of TAT still induced neurotoxicity while alcohol potentiated neuronal death, even at low doses.

Hemorrhage Associated Mechanisms of Neuroinflammation in Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major health problem for over 3.17 million people in the US, attracting increasing public attentions. Understanding the underlying mechanism of TBI is urgent for better diagnosis and treatment. Here, we examined the hypothesis that cerebral hemorrhagic coagulation and subsequent immune cells infiltration causes the progressive mechanisms of brain injury in moderate fluid percussion injury model. This represents a subdural hematoma and hemorrhagic head injury. We found increased hemorrhagic lesions and infarct volume in the injured brain with increment of pressure. The extent of hemorrhage was also validated by the bio-distribution of fluorescent tracer in cerebrospinal fluid (CSF) pathway after the injury. Bio-distribution of tracer was specifically diminished at the site of hemorrhage resulting from coagulation, which blocked the interstitial and CSF movement of the tracer. Increased expression of coagulation factor XII and necrotic cell death in and around the impact site confirmed the reason for this blockade. Different biomarkers, including immune cells accumulation and neuronal death showed that blood-brain barrier disruption played an important role for induction of neuroinflammation and neurodegeneration around the impact site. Our results suggest that instant hemorrhagic injury resulting from rupturing the brain blood vessels intertwined with coagulation causes onsite perivascular inflammation and neurodegeneration. Understanding of this sequential event should be valuable for development of therapeutic treatment in TBI. Graphical Abstract Underlying mechanisms in moderate/severe blunt TBI: hemorrhage following cerebrovascular disruption results in coagulation, thrombotic necrosis, and acute immune cell infiltration.

Primary blast causes mild, moderate, severe and lethal TBI with increasing blast overpressures: Experimental rat injury model.

Injury severity in blast induced Traumatic Brain Injury (bTBI) increases with blast overpressure (BOP) and impulse in dose-dependent manner. Pure primary blast waves were simulated in compressed gas shock-tubes in discrete increments. Present work demonstrates 24 hour survival of rats in 0-450 kPa (0-800 Pa∙s impulse) range at 10 discrete levels (60, 100, 130, 160, 190, 230, 250, 290, 350 and 420 kPa) and determines the mortality rate as a non-linear function of BOP. Using logistic regression model, predicted mortality rate (PMR) function was calculated, and used to establish TBI severities. We determined a BOP of 145 kPa as upper mild TBI threshold (5% PMR). Also we determined 146-220 kPa and 221-290 kPa levels as moderate and severe TBI based on 35%, and 70% PMR, respectively, while BOP above 290 kPa is lethal. Since there are no standards for animal bTBI injury severity, these thresholds need further refinements using histopathology, immunohistochemistry and behavior. Further, we specifically investigated mild TBI range (0-145 kPa) using physiological (heart rate), pathological (lung injury), immuno-histochemical (oxidative/nitrosative and blood-brain barrier markers) as well as blood borne biomarkers. With these additional data, we conclude that mild bTBI occurs in rats when the BOP is in the range of 85-145 kPa.

Interactions of oxidative stress and neurovascular inflammation in the pathogenesis of traumatic brain injury.

Traumatic brain injury (TBI) is a major cause of death in the young age group and leads to persisting neurological impairment in many of its victims. It may result in permanent functional deficits because of both primary and secondary damages. This review addresses the role of oxidative stress in TBI-mediated secondary damages by affecting the function of the vascular unit, changes in blood-brain barrier (BBB) permeability, posttraumatic edema formation, and modulation of various pathophysiological factors such as inflammatory factors and enzymes associated with trauma. Oxidative stress plays a major role in many pathophysiologic changes that occur after TBI. In fact, oxidative stress occurs when there is an impairment or inability to balance antioxidant production with reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels. ROS directly downregulate proteins of tight junctions and indirectly activate matrix metalloproteinases (MMPs) that contribute to open the BBB. Loosening of the vasculature and perivascular unit by oxidative stress-induced activation of MMPs and fluid channel aquaporins promotes vascular or cellular fluid edema, enhances leakiness of the BBB, and leads to progression of neuroinflammation. Likewise, oxidative stress activates directly the inflammatory cytokines and growth factors such as IL-1ß, tumor necrosis factor-α (TNF-α), and transforming growth factor-beta (TGF-ß) or indirectly by activating MMPs. In another pathway, oxidative stress-induced degradation of endothelial vascular endothelial growth factor receptor-2 (VEGFR-2) by MMPs leads to a subsequent elevation of cellular/serum VEGF level. The decrease in VEGFR-2 with a subsequent increase in VEGF-A level leads to apoptosis and neuroinflammation via the activation of caspase-1/3 and IL-1ß release.

Reduction of brain mitochondrial ß-oxidation impairs complex I and V in chronic alcohol intake: the underlying mechanism for neurodegeneration.

Neuropathy and neurocognitive deficits are common among chronic alcohol users, which are believed to be associated with mitochondrial dysfunction in the brain. The specific type of brain mitochondrial respiratory chain complexes (mRCC) that are adversely affected by alcohol abuse has not been studied. Thus, we examined the alterations of mRCC in freshly isolated mitochondria from mice brain that were pair-fed the ethanol (4% v/v) and control liquid diets for 7-8 weeks. We observed that alcohol intake severely reduced the levels of complex I and V. A reduction in complex I was associated with a decrease in carnitine palmitoyltransferase 1 (cPT1) and cPT2 levels. The mitochondrial outer (cPT1) and inner (cPT2) membrane transporter enzymes are specialized in acylation of fatty acid from outer to inner membrane of mitochondria for ATP production. Thus, our results showed that alterations of cPT1 and cPT2 paralleled a decrease ß-oxidation of palmitate and ATP production, suggesting that impairment of substrate entry step (complex I function) can cause a negative impact on ATP production (complex V function). Disruption of cPT1/cPT2 was accompanied by an increase in cytochrome C leakage, while reduction of complex I and V paralleled a decrease in depolarization of mitochondrial membrane potential (ΔΨ, monitored by JC-1 fluorescence) and ATP production in alcohol intake. We noted that acetyl-L-carnitine (ALC, a cofactor of cPT1 and cPT2) prevented the adverse effects of alcohol while coenzyme Q10 (CoQ10) was not very effective against alcohol insults. These results suggest that understanding the molecular, biochemical, and signaling mechanisms of the CNS mitochondrial ß-oxidation such as ALC can mitigate alcohol related neurological disorders.

Partial purification from hot dogs of N-nitroso compound precursors and their mutagenicity after nitrosation.

Hot dogs contain apparent N-nitroso compounds (ANC) and ANC precursors (ANCP). ANCP purification was followed by nitrosation, sulfamic acid treatment, and analysis for ANC. Aqueous hot dog extracts were adsorbed on silica gel, which was eluted with MeCN and MeOH. The MeOH eluate was adsorbed on cation exchange resin (H+ form) and eluted with NH4OH. Eluted ANCP traveled at moderate speeds in high-performance liquid chromatography (HPLC) on amino and Pb2+ columns. Gas chromatography-mass spectrometry (GC-MS) of trimethylsilyl (TMS) derivatives of crude water extract indicated the presence of glycerol, phosphate, lactic acid, and two monosaccharides. GC-MS of TMS derivatives of Pb2+ column HPLC eluates indicated that ANCP included 1-deoxy-N-1-glucosyl glycine. The nitrosated NH4OH eluate showed 4x background mutagenic activity for Salmonella typhimurium TA-100. Un-nitrosated fractions showed 2x background activity. Although tryptophan nitrosation gave 88% ANC yield, tryptophan is probably not a major ANCP in hot dogs. Hot dog patties prepared with or without sucrose or glucose showed similar ANC and ANCP levels. We discuss possible implications of these findings for the etiology of colon cancer.

Alcohol abuse enhances neuroinflammation and impairs immune responses in an animal model of human immunodeficiency virus-1 encephalitis.

Neuroinflammatory disorders (including human immunodeficiency virus-1 encephalitis, HIVE) are associated with oxidative stress and inflammatory brain injury, and excessive alcohol use can exacerbate tissue damage. Using a murine model of HIVE, we investigated the effects of alcohol abuse on the clearance of virus-infected macrophages and neuroinflammation. Severe combined immunodeficient mice were reconstituted with human lymphocytes, and encephalitis was induced by intracranial injection of HIV-1-infected monocyte-derived macrophages (HIV-1(+) MDM). Animals were fed an ethanol-containing diet beginning 2 weeks before lymphocyte engraftment and for the entire duration of the experiment. Lymphocyte engraftment was not altered by ethanol exposure. Alcohol-mediated immunosuppression in ethanol-fed mice was manifested by a significant decrease in CD8(+)/interferon-gamma(+) T lymphocytes, a fivefold increase in viremia, and diminished expression of immunoproteasomes in the spleen. Although both groups showed similar amounts of CD8(+) T-lymphocyte infiltration in brain areas containing HIV-1(+) MDMs, ethanol-fed mice featured double the amounts of HIV-1(+) MDMs in the brain compared to controls. Ethanol-exposed mice demonstrated higher microglial reaction and enhanced oxidative stress. Alcohol exposure impaired immune responses (increased viremia, decreased immunoproteasome levels, and prevented efficient elimination of HIV-1(+) MDMs) and enhanced neuroinflammation in HIVE mice. Thus, alcohol abuse could be a co-factor in progression of HIV-1 infection of the brain.

Blood-brain barrier: structural components and function under physiologic and pathologic conditions.

The blood-brain barrier (BBB) is the specialized system of brain microvascular endothelial cells (BMVEC) that shields the brain from toxic substances in the blood, supplies brain tissues with nutrients, and filters harmful compounds from the brain back to the bloodstream. The close interaction between BMVEC and other components of the neurovascular unit (astrocytes, pericytes, neurons, and basement membrane) ensures proper function of the central nervous system (CNS). Transport across the BBB is strictly limited through both physical (tight junctions) and metabolic barriers (enzymes, diverse transport systems). A functional polarity exists between the luminal and abluminal membrane surfaces of the BMVEC. As a result of restricted permeability, the BBB is a limiting factor for the delivery of therapeutic agents into the CNS. BBB breakdown or alterations in transport systems play an important role in the pathogenesis of many CNS diseases (HIV-1 encephalitis, Alzheimer's disease, ischemia, tumors, multiple sclerosis, and Parkinson's disease). Proinflammatory substances and specific disease-associated proteins often mediate such BBB dysfunction. Despite seemingly diverse underlying causes of BBB dysfunction, common intracellular pathways emerge for the regulation of the BBB structural and functional integrity. Better understanding of tight junction regulation and factors affecting transport systems will allow the development of therapeutics to improve the BBB function in health and disease.

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.

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.

Nitrosation of glycine ethyl ester and ethyl diazoacetate to give the alkylating agent and mutagen ethyl chloro(hydroximino)acetate.

Whereas nitrosation of secondary amines produces nitrosamines, amino acids with primary amino groups and glycine ethyl ester were reported to react with nitrite to give unidentified agents that alkylated 4-(p-nitrobenzyl)pyridine to produce purple dyes and be direct mutagens in the Ames test. We report here that treatment of glycine ethyl ester at 37 degrees C with excess nitrite acidified with HCl, followed by ether extraction, gave 30-40% yields of a product identified as ethyl chloro(hydroximino)acetate [ClC(=NOH)COOEt, ECHA] and a 9% yield of ethyl chloroacetate. The ECHA was identical to that synthesized by a known method from ethyl acetoacetate, strongly alkylated nitrobenzylpyridine, and may have arisen by N-nitrosation of glycine ethyl ester to give ethyl diazoacetate, which was C-nitrosated and reacted with chloride to give ECHA. Nitrosation of ethyl diazoacetate also yielded ECHA. Ethyl nitroacetate was not an intermediate as its nitrosation did not produce ECHA. ECHA reacted with aniline to give ethyl (hydroxamino)(phenylimino)acetate [PhN=C(NHOH)CO2Et]. This product was different from ethyl [(phenylamino)carbonyl]carbamate [PhNHC(=O)NHCO2Et], which was synthesized by reacting ethyl isocyanatoformate (OCN.CO2Et) with aniline. ECHA reacted with guanosine to give a derivative, which may have been a guanine-C(=NOH)CO2Et derivative. ECHA showed moderate toxicity and weak but significant mutagenicity without activation in Salmonella typhimurium TA-100 (mean, 1.31 x control value for 12-18 microg/plats) and for V79 mammalian cells (1.5-1.7 x control value for 60-100 microM). In conclusion, gastric nitrosation of glycine derivatives such as peptides with a N-terminal glycine might produce ECHA analogues that alkylate bases of gastric mucosal DNA and thereby initiate gastric cancer.

N-nitroso compounds in the gastrointestinal tract of rats and in the feces of mice with induced colitis or fed hot dogs or beef.

Because colonic N-nitroso compounds (NOC) may be a cause of colon cancer, we determined total NOC levels by Walters' method in the gastrointestinal tract and feces of rodents: (i) feces of C57BL mice fed chow and semi-purified diets contained 3.2 +/- 0.4 and 0.46 +/- 0.06 NOC/g, respectively (P < 0.01, mean +/- SD). (ii) NOC levels for gastrointestinal contents of three groups of Sprague-Dawley rats fed chow diet were 0.9 +/- 0.05 (diet), 0.2 +/- 0 (stomach), 0.3-0.4 (small intestine), 0.7-1.6 (cecum and colon) and 2.6 +/- 0.6 (feces) nmol/g. NOC precursor (NOCP) levels (measured as NOC after mild nitrosation) for two rat groups fed chow diet showed a 16-fold increase from stomach to proximal small intestine (mean, 6.2 micromol/g), and a 1.7-fold increase from distal colon to feces (mean, 11.6 micromol/g). (iii) Eight Min and five C57BL/6J mice received 4% dextran sulfate sodium in drinking water on days 1-4 to induce acute colitis. This increased fecal NOC levels 1.9-fold on day 5 in both strains (P < or = 0.04), probably due to NO synthase-derived nitrosating agents in the colon. (iv) Following studies on humans fed beef [Hughes et al. (2001) Carcinogenesis, 22, 199], Swiss mice received semi-purified diets mixed with 18% of beef plus pork hot dogs or sautéed beef for 7 days. On day 7, individual 24-h fecal NOC outputs were determined. In three hot dog and two beef groups with 5 mice/group, mean fecal NOC output/day was 3.7-5.0 (hot dog) and 2.0-2.9 (beef) times that for control groups fed semi-purified diet alone (P < 0.002 for each of combined groups). These groups showed little change in fecal NOCP output. (v) Initial purification of rat fecal NOCP by adsorption-desorption and HPLC is described. Results should help evaluate the view that colonic NOC causes colon cancer associated with colitis and ingestion of red and nitrite-preserved meat.

Total N-nitroso compounds and their precursors in hot dogs and in the gastrointestinal tract and feces of rats and mice: possible etiologic agents for colon cancer.

We review evidence that red and processed meat are causes of colon cancer and that processed meat is a risk factor for childhood cancer and type 2 diabetes. Associations could be due to N-nitroso compounds (NOCs) derived from nitrosation of NOC precursors (NOCPs). We review our survey of total NOC and NOCP content of foods. Only rapidly nitrosated amines, including a glycosyl amino acid, were efficiently determined as NOCPs. NOCPs in hot dogs and rat feces were partly purified by adsorption-desorption and HPLC. After nitrosation, purified hot dog fractions were directly mutagenic in Ames test. The main NOCPs in these materials may be N-glycosyl amino acids and peptides. NOC levels in rat gastrointestinal tract rose steadily from stomach to feces. NOCP levels showed similar trend but with sharp increases from stomach to duodenum. One day after Min and C57BL/6J mice were fed 4% dextran sulfate sodium to induce acute colitis, fecal NOC levels increased 1.9-fold compared with untreated mice (P < 0.05). For 7 d Swiss mice received semipurified diet, 180 g beef-pork hot dogs mixed with 820 g diet or 180 g sautéed beef mixed with 820 g diet. Fecal NOC outputs on day 7 were 3.7-5.0 (hot dog) and 2.0-2.9 (beef) times those for control groups (P < 0.002 for combined groups), perhaps reflecting higher dietary NOC intakes. Feeding a similar hot dog mixture to mice did not affect normal 7-methyldeoxyguanosine level in colonic mucosal DNA. Overall, results support the hypothesis that colonic NOCs are a cause of colon cancer.