Bilirubin as an Anti-oxidant for Surgical Stress: A Preliminary Report of Bilirubin Oxidative Metabolites

Background Bilirubin has been recognized as an antioxidant. The purpose of this study was to examine whether bilirubin would act as an antioxidant for surgical stress in humans. Materials and Methods Serum bilirubin and urinary bilirubin oxidative metabolites (BOM) were measured in 96 patients who underwent surgery. The antioxidant activity of bilirubin was assessed using BOM measured by enzyme-linked immunosorbent assay with an anti-bilirubin monoclonal antibody. Results Serum bilirubin levels increased after surgery in all 96 patients (p<0.01), but did not correlate with operation time or blood loss (p=0.53 and p=0.28, respectively). BOM increased only in patients with major surgeries (p=0.048). Significant correlations between BOM and operation time and blood loss were found (p<0.01). Conclusions Bilirubin appears to act as an antioxidant for invasive surgery in humans. Urinary BOM could be a reliable marker for the degree of surgical stress

Inverse gene expression patterns for macrophage activating hepatotoxicants and peroxisome proliferators in rat liver

Abstract Macrophage activation contributes to adverse effects produced by a number of hepatotoxic compounds. Transcriptional profiles elicited by two macrophage activators, LPS and zymosan A, were compared to those produced by 100 paradigm compounds (mostly hepatotoxicants) using cDNA microarrays. Several hepatotoxicants previously reported to activate liver macrophages produced transcriptional responses similar to LPS and zymosan, and these were used to construct a gene signature profile for macrophage activators in the liver. Measurement of cytokine mRNAs in the same liver samples by RT-PCR independently confirmed that these compounds are associated with macrophage activation. In addition to expected effects on acute phase proteins and metabolic pathways that are regulated by LPS and inflammation, a strong induction was observed for many endoplasmic reticulum-associated stress/chaperone proteins. Additionally, many genes in our macrophage activator signature profile were well-characterized PPARa-induced genes which were repressed by macrophage activators. A shared gene signature profile for peroxisome proliferators was determined using a training set of clofibrate, WY 14643, diethylhexylphthalate, diisononylphthalate, perfluorodecanoic acid, perfluoroheptanoic acid, and perfluorooctanoic acid. The signature profile included macrophage activator-induced genes that were repressed by peroxisome proliferators. NSAIDs comprised an interesting pharmacological class in that some compounds, notably diflunisal, co-clustered with peroxisome proliferators whereas several others co-clustered with macrophage activators, possibly due to endotoxin exposure secondary to their adverse effects on the gastrointestinal system. While much of these data confirmed findings from the literature, the transcriptional patterns detected using this toxicogenomics approach showed relationships between genes and biological pathways requiring complex analysis to be discerned.

Heme Oxygenase 1 expression after traumatic brain injury and effect of pharmacological manipulation on functional recovery.

Traumatic Brain Injury (TBI) is an increasingly diagnosed constellation of injuries derived from acute mechanical trauma to the brain. With the rise of advanced neuroimaging techniques recent focus has oriented primarily towards the mild-moderate range of TBI which previously was missed diagnostically. Characteristically, these advances have shown increasing areas of micro-hemorrhage in susceptible areas of the brain and to date there are no treatment modalities targeting micro-hemorrhages or their sequelae. This dissertation explores the effects of the resulting heme processing response in the days following injury with a particular focus on inducing early heme clearance from the parenchyma using a rat central fluid percussion injury model in the mild-moderate injury range. Since heme is released ~24-48 hours post-injury and is known to be cytotoxic we observed there may be a critical window for treatment to clear heme before it is spontaneously released and to increase the buffering capacity of the tissue. We targeted heme clearance by using drugs known to increased expression of Nrf2, an upstream transcriptional regulator of the canonical heme processing protein heme oxygenase 1 (HO-1), and tracking expression of HO-1, the iron sequestration/storage proteins Lipocalin 2 (LCN2) and Ferritin (FTL), as well as the activity of matrix metalloproteinases 2 and 9 (MMP2, MMP9). We examined both tissue known to be frankly hemorrhagic (the neocortex) as well as tissue lacking any identifiable bleed (the hippocampus). We demonstrated that using the HO-1 inducers Hemin and Sulforaphane in a single dose paradigm given 1 hour post-injury heme clearance was accelerated in the neocortex with the majority of heme pigment processed by 24 hours post-injury. Further there was significant attenuation of protein expression in HO-1 and ferritin as well as the enzyme activity of MMP2 and MMP9 in both the neocortex and the hippocampus. Behavioral attenuation was also seen in both rotarod and Morris water maze tests. While we intended to target hemorrhagic processing after injury, and indeed demonstrated improved clearance of heme from post-injury hemorrhagic regions of the brain, in both tissues studied we observed remarkably similar responses to the drugs utilized in protein expression, enzyme activity, and behavioral improvement which may suggest a globally improved pathologic state or that there are unidentified pathologic micro-hemorrhages or leaky vessels which extend further into the brain parenchyma than currently identified

Effects of heme oxygenase system on inflammation, oxidative stress, insulin signaling and tissue injury in liver, heart, kidney and pancreas in obese, diabetic and hypertensive rats

Tissue injury and metabolic dysfunction are salient features of obesity, diabetes, and hypertension. Inflammation and oxidative stress are considered key players behind this altered tissue homeostasis that drastically affect several vital organs including liver, heart, kidney and pancreas resulting in the development of hepatic steatosis, cardiomyopathy, nephropathy, pancreatic insulitis and abnormal glucose metabolism. Despite significant advancement in pharmaceutical interventions, the desired efficacy to manage these conditions is still lacking. In these situations, cytoprotective and functional attributes of the heme oxygenase (HO) system can be used as an alternative management tool. Although, the HO system is cytoprotective, its role in tissue injury and dysfunction in obesity, diabetes, and hypertension is not completely clear. To explore the importance of upregulating HO in these conditions, HO is induced through hemin in the rat models of obesity, diabetes and hypertension. My thesis work show that HO upregulation reduced the inflammation and oxidative stress-induced tissue injury in liver, heart, kidney, and pancreas through reduction of proinflammatory M1 macrophage marker (ED1) expression, cytokines (TNF-α, IL-1β, IL-6), oxidative-markers (8-isoprostane, ET-1) and profibrotic/extracellular matrix proteins (TGF-β, collagen-IV, fibronectin) and enhancement of the anti-inflammatory M2-macrophage markers (ED2, CD206, CD36, CD14), adiponectin and total-antioxidant capacity. HO upregulation improved glucose metabolism through potentiation of insulin signalling components (IRS-1, IRS-2, PI3K, GLUT4), reduced hyperglycemia, and enhanced several markers implicated in pancreatic repair and/or regeneration (c-Kit, Sca-1, Oct3/4, Pax2, β-catenin, Islet-1, Nkx6.1 and GLUT2). Collectively, the data from my thesis suggested the multifaceted cytoprotective mechanisms of the HO system against increasing tissue injury and metabolic dysfunction during obesity, diabetes and hypertension. Thus, HO upregulation through hemin may be part of therapeutic management strategies against tissue injury and metabolic dysfunction in obesity, diabetes and hypertension in the future

The handling of iron by erythroid and erythrophagocytic cells /

Iron is not a trace element in mammalian physiology. Using textbook values for blood volume (5.5 L), red blood cell (RBC) count (5 million/muL), and a lifespan of 120 days for red blood cells, the equilibrium value for the erythrocyte generation/death rate in the average adult male human is over 2 million/sec. It follows that the amount of iron required for hemoglobin synthesis in one day amounts to about 25 mg. Virtually every atom of that 25 mg is recycled by macrophages of the reticuloendothelial system (RES) that provide iron to the plasma for its subsequent delivery back to the erythron (with a small fraction going to other tissues). In light of the certain toxicity of unprotected iron, both erythroid precursors and RES macrophages perform remarkable tasks in handling such copious amounts of the catalytic metal. In my studies, I have examined specific aspects of iron metabolism in these two tissues.Iron is taken up by nearly every cell through a mechanism of receptor-mediated endocytosis, whereby the plasma iron binding protein transferrin (Tf) binds to its cognate receptor (TfR) on the cell surface, followed by internalization of the complex into a membrane bound organelle. Subsequent to endocytosis, the endosome is acidified by a v-ATPase proton pump, facilitating the release of iron from Tf. Through an unknown mechanism, iron is targeted to the inner membrane of the mitochondria, where the enzyme that inserts Fe2+ into protoporphyrin IX, ferrochelatase, resides. Although it has been demonstrated that the divalent metal transporter, DMT1, is responsible for the egress of reduced Fe from the vesicle, the immediate fate of the iron atoms after their transport across the vesicular membrane remains unknown. Therefore, we have investigated the uptake of iron in reticulocytes, cells that are taking up large amounts of iron for the synthesis of hemoglobin. Through both biochemical and imaging techniques, we have demonstrated that iron is transferred via a direct interorganellar relation between the endosome and mitochondria.The "haemoglobin-deficit" (hbd) mouse has an erythroid-specific mutation which is responsible for its microcytic, hypochromic phenotype. Previous studies have shown that these mice have normal dietary iron acquisition and normal to elevated serum iron levels. We therefore investigated the handling of iron in reticulocytes from these animals to determine whether the mutated gene possibly plays a role in the trafficking of transferrin-iron-containing organelles. A systematic examination of the steps in the transferrin pathway revealed that the intracellular trafficking of the protein is compromised in the hbd mice.The rapid turnover of iron by macrophages of the RES requires heme oxygenase-1 (HO-1), which catalyzes the rate-limiting step in heme degradation. This highly inducible enzyme, besides its major role in erythrocyte iron recycling, has been demonstrated to confer astonishing cytoprotectivity to cells and tissues in which its expression is elevated (either through chemical induction or genetic manipulation). In addition to, reportedly protective, carbon monoxide and biliverdin, the HO-1 catalyzed reaction releases ferrous iron, which itself is a potent pro-oxidant. Also, it is unlikely that there exists a significant amount of free heme in most tissues (i.e., non-erythroid, non-erythrophagocytic), to provide significant amounts of substrate to this enzyme. Hence, it is tempting to speculate that the mechanism of heme oxygenase cytoprotection is removed from its function of heme catabolism. Therefore, we investigated whether increased expression of heme oxygenase will, in and of itself, alter iron metabolism in cultured cells. My experiments show that in the absence of exogenous hemin, elevation of HO-1 protein levels does not have any effect on cellular iron metabolism in cultured cells

Invertebrate stress responses as molecular biomarkers in ecotoxicology

All organisms studied so far respond to heat shock by inducing the synthesis of a number of proteins called heat shock proteins(LISPs). This universal response can also be induced by a variety of stressors, including heavy metal ions and organic and organo-metallic compounds. As a result, the stress response has recently attracted the attention of ecotoxicologists for use in environmental biomonitoring. In the present study, we have investigated the stress responses of two different organisms ; namely the free-living soil nematode Caenorhiabdities elegans(both wild-type and transgenic strains) and the freshwater crustacea Asellus aquazicus. We have also explored the possible use of these model systems in environmental monitoring using different techniques which include metabolic labelling with subsequent one-dimensional electrophoresis and autoradiography, and one- or two-dimensional western blotting using antibodies specific to stress protein 70. The study with A. aquaticus shows that this organism exhibits a classical stress response. The exposure of asellids to heat shock-treatment (26°C ; 13°C above the standard maintenance temperature) or to sublethal concentrations of metal ions (Cd++ and Cu++) resulted in the induction of at least 5 putative HSPs which belong to several major HSP families (HSP100, HSP90 and possibly HSP60). An increase in the synthesis of smaller sizes of polypeptides (25-35 kD) should be also noted. Moreover, the time-course of heat versus heavy metal stress-response in this organism suggests that the pattern of stress-protein synthesis changes considerably with increasing exposure time ; notably the response to heat is more transient than that to heavy metals. However, HSP70 does not appear to be the major stress protein induced in this organism. The presence of low molecular weight (LMW) proteins which react with anti-HSP70 antibodies and the apparent deficiency of classical 70 kD stress proteins in A. aquaticus, both suggest that HSP70s in this organism are for some reason prone to degradation. In the nematode C .elegans, shifting the culture temperature from 20°C to 34°C induces the synthesis of a set of HSPs corresponding to the HSP90, HSP70 and small HSP families. There are at least nine members of the hsp7O multigene family in C. elegans ; some members are expressed constitutively while others are stress inducible. W e have studied the effects of heat and heavy metal (cadmium) stress on the expression patterns of the HSP70 protein family in the nematodes by one- and two-dimensional Western blotting using a monoclonal anti-HSP70 antibody that recognises a conserved epitope shared by most HSP70 family members. Constitutive C. elegans HSP70s (expressed at 20°C) are almost undetectable on one-dimensional immunoblots, but chemiluminescent probing of two-dimensional blots reveals a complex pattern of several HSP70s pots .Mild heats hock at 31° C induces a doublet HSP70 band on one-dimensional blots, of which the heavier component (75 kD) is more prominent than the lighter (73 kD). On two-dimensional blots, this pattern is shown to be more complex with a prominent 75 kD spot newly induced and several other spots intensified. Severe heat shock at 34°C strongly induces both 75 and 73 kD bands on one-dimensional blots; two dimensional analysis reveals a series of novel and/or elevated 73 and 75 kD spots. Treatment with cadmium( 16 ppm) at 31° C gives a different pattern of spots as compared with 31 °C alone ; several spots show enhanced while some are newly expressed, and not all of these are present at 34°C. These results indicate that related members of the HSP70 protein family in C. elegans are independently regulated in response to different forms of stress. The possible significance of these findings is discussed in relation to the possible use of stress responses s environmental biomonitors. We have also utilised a stress-inducible C. elegans strain (CB4027) for monitoring environmental contamination. This transgenic strain carries integrated copies of the Drosophila hsp70 promoter fused to an E.coli lacZ reporter gene. When exposed to heat shock or to several environmentally relevant stressors, the transgenic strain expresses the reporter product, 3-galactosidase, which can easily be quantified or localised in situ in stained worms or on Western blots (apparently enzymatically active as a 170 kD form). We have exposed transgenic worms to a variety of toxicants at an elevated temperature (32°C) just below that required for heat shock (34°C), in order to obtain optimal transgene induction. Exposure of nematodes to several heavy metals (e. g. Cd+, Hg++, Zn+, Sn++, Mn++ and Ag+), organometallic toxicants (tributyltin) or organic pollutants (lindane) induces ß-galactosidase expression in a dose-dependent manner. Cadmium is found to be by far the strongest inducer of transgene activity amongst the agents tested, although tributyltin is an effective inducer at ppb levels. The effects of mixtures of divalent metal ions (Cd++/Ca++, Cd++/Zn++ and Cd++/Hg++) on ß-galactosidase expression have been also investigated. All three divalent ions tested in combination with cadmium significantly inhibit cadmium-induced transgene activity in comparison to cadmium alone. In the case of Cd++/C++ mixtures, a marked inhibition of Cd++ accumulation by worm tissues has also been demonstrated, directly related to the Ca++ concentration. These effects may represent competition for metal-ion uptake through calcium channels. Our results show that this transgenic system works well within strictly defined assay conditions, and can detect clear responses over a 7h exposure period to environmentally relevant toxicants at sublethal concentrations well below the 24 or 48h LCSO values. However, there is a need for careful characterisation and containment of any transgenic organism if it is to be used as environmental monitoring tool