Bilirubin Formation Research Articles

Effect of light exposure on metalloporphyrin-treated newborn mice

Neonatal hyperbilirubinemia arises from increased bilirubin production and decreased bilirubin elimination. Although phototherapy safely and effectively reduces bilirubin levels, recent evidence shows that it has adverse effects. Therefore, alternative treatments are warranted. Metalloporphyrins, competitive inhibitors of heme oxygenase (HO), the rate-limiting enzyme in bilirubin production, effectively reduce bilirubin formation; however, many are photoreactive. Here, we investigated possible photosensitizing effects of chromium mesoporphyrin (CrMP) and zinc deuteroporphyrin bis-glycol (ZnBG). Administration of CrMP or ZnBG to 3-d-old mouse pups (3.75-30.0 μmol/kg intraperitoneally) and exposure to cool white (F20T12CW) and blue (TL20W/52) fluorescent lights (+L) for 3 h, resulted in a dose-dependent mortality (50% lethal dose (LD50) = 21.5 and 19.5 μmol/kg, respectively). In contrast to ZnBG, there was no significant difference in survival between the CrMP+L and CrMP groups. Following 30 μmol/kg ZnBG+L, we found significant weight loss, decreased liver antioxidant capacities, and increased aspartate aminotransaminase levels. At 6-d post-light exposure, ZnBG+L-treated pups showed gross and histologic skin changes at doses >7.5 μmol/kg. No lethality was observed following treatment with 30 μmol ZnBG/kg plus exposure to blue light-emitting diodes. Phototoxicity of ZnBG was dependent on light source, emission spectrum, and irradiance. Low doses of ZnBG (<3.75 μmol/kg) retained maximal HO inhibitory potency without photosensitizing effects, and therefore are potentially useful in treating neonatal hyperbilirubinemia.

Cigarette smoke extract upregulates heme oxygenase-1 via PKC/NADPH oxidase/ROS/PDGFR/PI3K/Akt pathway in mouse brain endothelial cells

BackgroundIn the brain, the inducible form of heme oxygenase (HO-1) has been recently demonstrated to exacerbate early brain injury produced by intracerebral hemorrhagic stroke which incident rate has been correlated with cigarette smoking previously. Interestingly, cigarette smoke (CS) or chemicals present in CS have been shown to induce HO-1 expression in various cell types, including cerebral endothelial cells. However, the mechanisms underlying CS modulating HO-1 protein expression are not completely understood in the brain vessels.ObjectiveThe aim of the present study was to investigate the mechanisms underlying CS modulating HO-1 protein expression in cerebral endothelial cells.MethodsCultured cerebral endothelial cells (bEnd.3) were used to investigate whether a particulate phase of cigarette smoke extract (PPCSE) regulates HO-1 expression and to investigate the molecular mechanisms involved in HO-1 expression in bEnd.3 cells.ResultsWe demonstrated that PPCSE (30 μg/ml) significantly induced HO-1 protein expression and its enzymatic activity in bEnd.3 cells determined by western blotting and bilirubin formation, respectively. PPCSE-induced HO-1 expression was mediated through phosphatidylcholine phospholipase C (PC-PLC), PKCδ, and PI3K/Akt which were observed by pretreatment with their respective pharmacological inhibitors or transfection with dominant negative mutants of PKCδ and Akt. ROS scavenger (N-acetyl-L-cysteine, NAC) blocked the PPCSE-induced ROS generation and HO-1 expression. Pretreatment with selective inhibitors of PKCδ (rottlerin) and NADPH oxidase [diphenyleneiodonium chloride (DPI) and apocynin (APO)] attenuated the PPCSE-induced NADPH oxidase activity, ROS generation, and HO-1 expression. In addition, we found that PPCSE induced PI3K/Akt activation via NADPH oxidase/ROS-dependent PDGFR phosphorylation.ConclusionsTaken together, these results suggested that PPCSE-induced HO-1 expression is mediated by a PC-PLC/PKCδ/NADPH oxidase-dependent PDGFR/PI3K/Akt pathway in bEnd.3 cells.

Adult jaundice—the pathophysiology, classification and causes

Jaundice (icterus) is the commonest presentation in patients with liver disease, and is caused by excessive bilirubin (&gt;17μmol/litre) leading to deposits in subcutaneous tissue e.g. skin, sclera and mucous membranes. Jaundice can be confirmed by a number of simple investigations including patient history, physical examination and biochemical screening; however, the cause of the jaundice may require further investigation. In this article, Suzanne Sargent and Michelle Clayton provide an overview of the pathophysiology of the formation and metabolism of bilirubin and examines the most common causes of jaundice in the adult patient.

In vitro inhibition of heme oxygenase isoenzymes by metalloporphyrins

Neonatal jaundice results from an increased bilirubin production and decreased hepatic bilirubin conjugation and excretion. Severe hyperbilirubinemia is currently treated with phototherapy or exchange transfusion; however, its prevention by inhibiting bilirubin formation is a more logical strategy. Heme oxygenase (HO), with inducible (HO-1) and constitutive (HO-2) isoenzymes, is the rate-limiting enzyme in heme catabolism, producing equimolar amounts of bilirubin and carbon monoxide (CO). Metalloporphyrins (Mps) are heme derivatives that competitively inhibit HO and thereby suppress hyperbilirubinemia. No systematic studies have been reported evaluating whether the HO isoenzymes are inhibited differentially by various Mps. Identification of Mps that selectively inhibit the inducible HO-1 without affecting the 'housekeeping' HO-2 isoenzyme might be desirable in the clinical setting of hemolytic disease, in which the Hmox1 gene is greatly induced. Although bilirubin production is due to the activity of both HO-1 and HO-2, the inhibition of HO-1 with a relative sparing of HO-2 activity might provide the most selective approach for the treatment of hemolytic disease. We determined for the deutero-, proto-, meso- and bis-glycol porphyrins with zinc, tin and chromium as central atoms, respectively, the concentration needed for 50% inhibition (I(50)) of HO-1 and HO-2 activities in rat spleen and brain tissue. For a given Mp, HO-1 activity was less inhibited than that of HO-2. The order of inhibitor potency of each Mp was nearly identical for both isoenzymes. Tin mesoporphyrin was the most potent inhibitor for both isoenzymes. HO-2 selectivity was greatest for tin protoporphyrin. Conversely, the Zn compounds were least inhibitory toward HO-2. No Mp preferentially inhibited HO-1. Mps that produce a less inhibitory effect on HO-2, while limiting the response of the inducible HO-1, such as ZnPP, may be a useful clinical tool.

The role of HO-CO system in the hemodynamic disturbance of cirrhotic rats

To investigate the role of heme oxygenase(HO), a catalyzing enzyme of heme to produce CO, in modulation of systemic circulation in CCl4-induced cirrhotic rats. Saline(vehicle) and ZnPP were s.c. injected into the posterior necks of rats respectively and the rats were then anesthetized by pentobarbital sodium in four hours. Mean arterial pressure (MAP, kPa), heart rate (HR, b/min) and portal pressure (PP, cm/H2O) were measured by indwelling catheter. Plasma CO was determined by Chalmers method. Heme oxygenase acivity was determined by the rate of bilirubin formation. The cirrhotic rats showed significant hyperdynamic circulation indicated by decreased mean arterial pressure [MAP, (15.6+/-1.7) vs (18.9+/-0.9) kPa, t = 4.52, P less than 0.01] and increased portal pressure [PP, (16.7+/-0.8) vs (8.8+/-0.3) cm H2O, t = 23.10, P less than 0.01] as compared to normal control rats(NS). ZnPP could cause a significant increase in MAP [(17.3+/-1.5) vs (15.6+/-1.7) kPa, t = 2.18, P less than 0.05] and significant decrease in PP [(13.2+/-0.7) vs (16.7+/-0.8) cm H2O, t = 8.53, P less than 0.01] in cirrhotic rats. The cirrhotic group presented a significant increase in plasma CO [(18.0+/-1.9) vs (10.4+/-1.3)mumol/L, t = 8.42, P less than 0.01] and HO activity in the spleens [(11.1+/-0.9) vs (6.5+/-0.9) nmol bilirubin/mg protein/h, t = 9.28, P less than 0.01] and intestines [(2.5+/-0.1) vs. (1.3+/-0.2) nmol bilirubin/mg protein/h, t = 15.1, P less than 0.01]. ZnPP could cause significant decreases in plasma CO and HO activity in liver, spleen and intestine of both control and cirrhotic rats. HO-CO system activation may be an important reason for the hemodynamic disturbance of liver cirrhosis.

Circular Dichroism Spectroscopy Is a Sensitive Tool for Investigation of Bilirubin−Enzyme Interactions

Noncovalent complex formation of unconjugated bilirubin with various enzymes has been demonstrated by measuring induced circular dichroism (ICD) peaks associated with the pigment VIS absorption band. Preferential binding of the P- or M-helical conformer of bilirubin to dehydrogenases, catalase, alkaline phosphatase, and α-chymotrypsin is responsible for the characteristic exciton CD couplet that undergoes remarkable changes upon the addition of enzymatic cofactors (NADH, AMP) and an inhibitor (acridine). Alterations of the ICD spectra refer to a direct binding competition between bilirubin and NADH for a common binding site on alcohol dehydrogenase and catalase, suggesting a potential mechanism for the inhibitory effect of BR reported on NAD(P)H dependent enzymes. NADH and bilirubin form a ternary complex with glutamate dehydrogenase indicated by peculiar CD spectral changes that are proposed to be generated by allosteric mechanism. α-chymotrypsin binds bilirubin in its catalytic site, as indicated by CD displacement experiments performed with the competitive inhibitor acridine. Surprisingly, the closely related trypsin does not induce any CD signal with bilirubin. Taking into consideration the clinically relevant but controversial and poorly understood areas of bilirubin biochemistry, the fast and simple CD spectroscopic approach presented here may help to unfold diverse physiological and pathophysiological roles of BR on a molecular level.

Conversion of biliverdin to bilirubin by biliverdin reductase contributes to endothelial cell protection by heme oxygenase-1—evidence for direct and indirect antioxidant actions of bilirubin

Heme oxygenase-1 (HO-1) is highly protective in various pathophysiological states such as cardiovascular and neurodegenerative diseases. HO-1-derived bilirubin is an efficient scavenger of reactive oxygen and nitrogen species (RONS). It remains to determine whether conversion of biliverdin to bilirubin is an essential step for HO-1-conferred protection of endothelial cells. RONS scavenging activities of biliverdin versus bilirubin were assessed by different RONS generating systems and detection techniques. We also silenced the biliverdin reductase (BVR) or HO-1 gene in cultured primary human endothelial cells (HUVECs) and measured the effect on RONS formation upon stimulation with lipopolysaccharide (LPS). In addition, effects of bilirubin and biliverdin on expression of GTP-cyclohydrolase were assessed in an endothelial cell line (EA.hy 926). HO-1- and BVR-silenced cells have increased levels of oxidative stress and bilirubin but not biliverdin increased expression of the protective protein GTP-cyclohydrolase. Moreover, protection by hemin-induced HO-1 expression or biliverdin-triggered bilirubin formation was impaired upon silencing of the HO-1 or BVR gene, respectively. Since bilirubin significantly scavenged RONS but chronic treatment was even more protective our observations support direct and indirect antioxidant properties of BVR and bilirubin and an important role for BVR and bilirubin in HO-1 conferred protection of endothelial cells.

A Professional and Personal Odyssey

As I began to write this article reflecting on my professional career in biomedical research, many memories of people, activities, situations, and experiences were swirling in my head. Because of the extraordinary support that I have received from my family, they are never far from my thoughts. I believe that sharing a few of these memories will be helpful for those who find themselves in similar situations or periods of professional development. This is not meant to be a comprehensive review of the fields to which I have had the privilege of contributing but a personal stroll down a path in which I have found extraordinary pleasure and from which I have derived a sense of accomplishment.

Measurement of Membrane‐Bound Human Heme Oxygenase‐1 Activity Using a Chemically Defined Assay System

Heme oxygenase (HO) catalyzes heme degradation in a reaction requiring NADPH‐cytochrome P450 reductase (CPR). Although most studies with HO used a 30‐kDa form, lacking the C‐terminal membrane binding region, the catalytic behavior of this enzyme is very different if this domain is retained; the overall activity being elevated 5‐fold, and the Km for CPR decreased approximately 50‐fold. The goal of these studies was to accurately measure membrane‐bound heme oxygenase‐1 (HO‐1) activity using a coupled assay containing purified biliverdin reductase (BVR). When rat liver cytosol was used as the source of partially purified BVR, the reaction remained linear for 2‐3 min; however, the reaction was only linear for 10‐30 sec when an equivalent amount of purified, human BVR (hBVR) was used. This lack of linearity was not observed with soluble HO‐1. Optimal formation of bilirubin was achieved with concentrations of bovine serum albumin (0.25 mg/ml) and hBVR (0.025‐0.05 µM), but neither supplement increased the time that the reaction remained linear. Superoxide dismutase had no effect on the reaction; however, when catalase was included, the reactions were linear for at least 4‐5 minutes, even at high CPR levels. These results not only demonstrate that hydrogen peroxide leads to a decrease in HO‐1 activity, but also provide a chemically defined system to further examine the function of full‐length HO‐1 in a membrane environment.

Measurement of Membrane-Bound Human Heme Oxygenase-1 Activity Using a Chemically Defined Assay System

Heme oxygenase (HO) catalyzes heme degradation in a reaction requiring NADPH-cytochrome P450 reductase (CPR). Although most studies with HO used a soluble 30-kDa form, lacking the C-terminal membrane-binding region, recent reports show that the catalytic behavior of this enzyme is very different if this domain is retained; the overall activity was elevated 5-fold, and the K(m) for CPR decreased approximately 50-fold. The goal of these studies was to accurately measure HO activity using a coupled assay containing purified biliverdin reductase (BVR). This allows measurement of bilirubin formation after incorporation of full-length CPR and heme oxygenase-1 (HO-1) into a membrane environment. When rat liver cytosol was used as the source of partially purified BVR, the reaction remained linear for 2 to 3 min; however, the reaction was only linear for 10 to 30 s when an equivalent amount of purified, human BVR (hBVR) was used. This lack of linearity was not observed with soluble HO-1. Optimal formation of bilirubin was achieved with concentrations of bovine serum albumin (0.25 mg/ml) and hBVR (0.025-0.05 microM), but neither supplement increased the time that the reaction remained linear. Various concentrations of superoxide dismutase had no effect on the reaction; however, when catalase was included, the reactions were linear for at least 4 to 5 min, even at high CPR levels. These results not only show that HO-1-generated hydrogen peroxide leads to a decrease in HO-1 activity but also provide for a chemically defined system to be used to examine the function of full-length HO-1 in a membrane environment.

Preparation of chitosan-polyaspartic acid-5-fluorouracil nanoparticles and its anti-carcinoma effect on tumor growth in nude mice

To prepare chitosan-polyaspartic acid-5-fluorouracil (CTS-Pasp-5Fu) nanoparticles and investigate its anti-carcinoma effect and toxicity. CTS-Pasp-5Fu nanoparticles were synthesized by ionic gelatification. Male BABL/c nude mice were injected with SGC-7901 gastric carcinoma cell line mass to establish a human gastric carcinoma model. They were randomly allocated into 4 groups: CTS-Pasp-5Fu (containing 5-Fu 1.25 mg/kg), 5-Fu (1.25 mg/kg), CTS-Pasp and normal saline groups. Tumor weight was measured and assay of colony forming unit-granulocyte and macrophage (CFU-GM) was performed. The structural change of cells and tissues was observed and the Bax and Bcl-2 genes were detected. Compared with normal saline, the inhibition rates of tumor growth for the CTS-Pasp, 5-Fu and CTS-Pasp-5Fu groups were 5.58%, 58.69% and 70.82%, respectively. The tumor inhibition rates for the CTS-Pasp, 5-Fu and CTS-Pasp-5Fu groups were 5.09%, 65.3% and 72.79%, respectively. There was a significant decrease in the number of CFU-GM formation and increase of total bilirubin, and alanine aminotransferase in the 5-Fu group, but no change in those of the other three groups. There was no change in white blood cell count and creatinine among the four groups. Pathological section of liver and nephridial tissues showed that the damage in the 5-Fu group was more severe than that in the CTS-Pasp-5Fu group. 5-Fu and CTS-Pasp-5Fu groups could both down-regulate the Bcl-2 expression and up-regulate the Bax expression to different extent, and the accommodate effect of CTS-Pasp-5Fu was more obvious than 5-Fu. The tumor inhibition rate of CTS-Pasp-5Fu nanoparticles is much higher than that of 5-Fu alone.

Trans-Arachidonic acids induce a heme oxygenase-dependent vasorelaxation of cerebral microvasculature

Nitrative stress is an important regulator of vascular tone. We have recently described that trans-arachidonic acids ( TAA) are major products of NO 2 ·–mediated isomerization of arachidonic acid in cell membranes and that nitrative stress increases TAA levels leading to neural microvascular degeneration. In the present study, we explored whether TAA exert acute effects on neuromicrovascular tone and investigated potential mechanisms thereof. TAA induced an endothelium-dependent vasorelaxation of rat brain pial microvasculature. This vasorelaxation was independent of nitric oxide, prostanoids, lipoxygenase products, and CYP 450 metabolite trans-hydroxyeicosatetraenoic acids. However, inhibition of heme oxygenase (using zinc protoporphyrin IX) and of dependent soluble guanylate cyclase (sGC; using ODQ) significantly diminished (by ∼ 70%) the TAA-induced vasorelaxation. Consistent with these findings, TAA stimulated heme oxygenase (HO)-2-dependent bilirubin (using siRNA HO-2) and cGMP formation, and the HO product carbon monoxide (using CO-releasing CORM-2) reproduced the sGC-dependent cGMP formation and vasorelaxation. Further exploration revealed that TAA-induced vasorelaxation and bilirubin formation (HO activation) were nearly abrogated by large-conductance calcium-dependent potassium channels (BK Ca) (using TEA and iberiotoxin). Opening of BK Ca with the selective activator NS1619 induced a concentration-dependent vasorelaxation, which was inhibited by HO and sGC inhibitors. Coimmunoprecipitation suggested a molecular complex interaction between BK Ca and HO-2 (but not HO-1). Collectively, these findings identify new properties of TAA, specifically cerebral vasorelaxation through interactive activation of BK Ca with HO-2 and, in turn, sGC. Our findings provide new insights into the characterization of nitrative stress-derived TAA products, by showing they can act as acute mediators of nitrative stress on neurovascular tone.

Statin treatment increases formation of carbon monoxide and bilirubin in mice: a novel mechanism of in vivo antioxidant protection

Heme oxygenase (HO) has a central role in cellular antioxidant defences and vascular protection, and it may mediate pleiotropic actions of drugs used in cardiovascular therapy. We investigated whether long-term use of statins upregulates HO activity and increases carbon monoxide (CO) and bilirubin levels in vivo. Adult FvB mice were given atorvastatin or rosuvastatin (5 mg/kg) daily by i.p. injections for 1, 2, or 3 weeks. HO activity, tissue CO, bilirubin, and antioxidant levels, total plasma bilirubin, and carboxyhemoglobin (COHb) were measured. Fold changes in heart HO activity significantly increased after 1, 2, and 3 weeks of atorvastatin (1.24 +/- 0.06 (p < or = 0.05); 1.29 +/- 0.26 (p < or = 0.03); 1.33 +/- 0.08 (p < 0.01), respectively) and 2 and 3 weeks of rosuvastatin (1.23 +/- 0.20 (p < or = 0.03); 1.63 +/- 0.42 (p < 0.01), respectively). Heart tissue CO and COHb levels also increased after 3 weeks with atorvastatin (1.30 +/- 0.24 (p < or = 0.05); 1.92 +/- 0.17 (p < or = 0.001), respectively) and rosuvastatin (1.47 +/- 0.13 (p < or = 0.004); 1.63 +/- 0.12 (p < or = 0.001), respectively). Significant increases in heart antioxidant levels were observed after statin treatment and corroborated by heart bilirubin content elevations. Antioxidant level increases were abolished by treatment with an HO inhibitor. These findings suggest that the induction of HO and the production of its products, CO and bilirubin, may be a mechanism by which statins exert antioxidant actions and confer cardioprotection in vivo.

Regulation of TNF‐α‐activated PKC‐ζ signaling by the human biliverdin reductase: identification of activating and inhibitory domains of the reductase

Human biliverdin reductase (hBVR) is a dual function enzyme: a catalyst for bilirubin formation and a S/T/Y kinase that shares activators with protein kinase C (PKC) -zeta, including cytokines, insulin, and reactive oxygen species (ROS). Presently, we show that hBVR increases PKC-zeta autophosphorylation, stimulation by TNF-alpha, as well as cytokine stimulation of NF-kappaB DNA binding and promoter activity. S149 in hBVR S/T kinase domain and S230 in YLS230F in hBVR's docking site for the SH2 domain of signaling proteins are phosphorylation targets of PKC-zeta. Two hBVR-based peptides, KRNRYLS230F (#1) and KKRILHC281 (#2), but not their S-->A or C-->A derivatives, respectively, blocked PKC-zeta stimulation by TNF-alpha and its membrane translocation. The C-terminal-based peptide KYCCSRK296 (#3), enhanced PKC-zeta stimulation by TNF-alpha; for this, Lys296 was essential. In metabolically 32P-labeled HEK293 cells transfected with hBVR or PKC-zeta, TNF-alpha increased hBVR phosphorylation. TNF-alpha did not stimulate PKC-zeta in cells infected with small interfering RNA for hBVR or transfected with hBVR with a point mutation in the nucleotide-binding loop (G17), S149, or S230; this was similar to the response of "kinase-dead" PKC-zeta(K281R). We suggest peptide #1 blocks PKC-zeta-docking site interaction, peptide #2 disrupts function of the PKC-zeta C1 domain, and peptide #3 alters ATP presentation to the kinase. The findings are of potential significance for development of modulators of PKC-zeta activity and cellular response to cytokines.

The effect of the initial reactant molar ratio and doping with Fe3+ on the formation of calcium bilirubinate in water-oil microemulsions

A series of calcium bilirubinate nanoparticles were synthesized in microemulsions consisting of polyoxyethylene octylphenol ether (Triton X-100), n-hexyl alcohol (n-C6H13OH), cyclohexane (c-C6H12), and an aqueous solution. The particles were characterized by Fourier transform infrared (FT-IR) spectra, powder X-ray diffraction (XRD), inductively coupled plasma atomic emission spectrometry (ICP-AES), transmission electron microscopy (TEM), and zeta potential measurements. The results showed that the initial CaCl2/Na2BR molar ratio and the molar ratio of water to surfactant (ω0) influenced the morphology and microstructure of calcium bilirubinate nanoparticles and the sites of Ca2+ coordination to BR2−. The composition of synthesized calcium bilirubinate was nonstoichiometric. The stability of calcium bilirubinate nanoparticles dispersed in water changes as the Ca2+/BR2− molar ratio and solution pH varied. Dopant Fe2+ ions played a certain role in the simulated mineralization of calcium bilirubinate. A possible mechanism of calcium bilirubinate formation in inverse microemulsions is discussed.

Gallstone disease: Free radical reactions and the ambivalent role of bilirubin in the pathomechanism of gallstone formation

In the formation of gallstone diseases, there are numerous genes, that are held responsible for liver disorders primarily and diseases of bile flow and bile formation, modification of lipid metabolism diabetes mellitus, obesity, glutene sensitive enteropathy, Crohn-disease, Down syndrome, Gucher syndrome, cystic fibrosis as well as haematological disorders and state following ileum resection. In the development of these, bacterial infections, inflammatory reactions, metal element power and free radicals play an important role. Fatty acids, lipid oxides, diene conjugates and other lipid peroxidation products are moving from the liver to the bile, and they initiate primer and secondary free radical reactions in the bile duct and gallbladder. The inflammation processes in the gallbladder wall produce free radicals. The free bilirubin content of the bile behaves pro- and antioxidant molecules. The ambivalent property of free bilirubin, which is detected concentration in the gallbladder bile -- from microsomal leakage or as a consequence of bacterial deglucuronidation -- increases the free radical reactions in the gallbladder. The gallstone formation of free bilirubin with metal ions, primarily Ca ++ ions makes calcium hydrogen bilirubinate in the bile. Calcium ions can react with fatty acids and hereby modify the bile viscosity. The lipids, free bilirubin and metal elements are all components in stone formation. Antioxidants, concerning their derivates or molecules, medicines, which increase antioxidant property can influence the bile composition or inhibit the gallstone formation on several levels.

Reduction of bilirubin by targeting human heme oxygenase-1 through siRNA.

Neonatal hyperbilirubinemia is a common clinical condition caused mainly by the increased production and decreased excretion of bilirubin. Current treatment is aimed at reducing the serum levels of bilirubin. Heme oxygenase-1 (HO-1) is a rate-limiting enzyme that generates bilirubin. In this study we intended to suppress HO-1 using the RNA interference technique. Small interfering RNA (siRNA)-A, -B, and -C were designed based on human HO-1 (hHO-1) mRNA sequences. siRNA was transfected into a human hepatic cell line (HL-7702). hHO-1 transcription and protein levels were then determined. In addition, the inhibitory effect of siRNA on hHO-1 was assessed in cells treated with hemin or transfected with an hHO-1 plasmid. siRNA-C showed the most potent suppressive effect on hHO-1. This inhibition is dose and time dependent. Compared with control, both hemin and hHO-1 plasmids up-regulated hHO-1 expression in HL-7702 cells. However, the up-regulation was significantly attenuated by siRNA-C. Furthermore, the decrease in hHO-1 activity was coincident with the suppression of its transcription. Finally, siRNA-C was shown to reduce hHO-1 enzymatic activity and bilirubin levels. Thus, this study provides a novel therapeutic rationale by blocking bilirubin formation via siRNA for preventing and treating neonatal hyperbilirubinemia and bilirubin encephalopathy at an early clinical stage.

Modulation by heme and zinc protoporphyrin of colonic heme oxygenase-1 and experimental inflammatory bowel disease in the rat

Reactive oxygen species, suggested to be involved in inflammatory bowel disease, may be modulated by endogenous anti-oxidant products of heme oxygenase-1 (HO-1). In the present work, HO-1 expression in trinitrobenzene sulphonic acid (TNBS)-induced colitis in the rat and the effects of HO-1 modulation, particularly by the HO-1 inducer, heme, were further evaluated. Colitis was induced by intracolonic challenge with TNBS and assessed macroscopically and by myeloperoxidase (MPO) assay. Heme oxygenase activity was determined by measurement of bilirubin formation and HO-1 protein expression was determined by Western blotting. TNBS challenge led to an early and substantial induction of HO-1 protein expression and heme oxygenase activity in the colon that peaked after 48–72 h and declined over 10 days. Heme (30 μmol/kg/day, s.c) increased colonic HO-1 protein expression and enzyme activity and decreased colonic damage and myeloperoxidase activity. Short-term administration of cadmium chloride (2 mg/kg, s.c.), another known HO-1 inducer, also reduced the colonic injury and myeloperoxidase levels. In contrast, the HO-1 inhibitor, zinc protoporphyrin (50 μmol/kg/day, s.c) significantly increased the colonic damage and myeloperoxidase activity over 10 days, as did tin protoporphyrin (30 μmol/kg/day, s.c). These results support the proposal that induction of HO-1 provides a protective mechanism in this model under both acute and more-chronic conditions, and that its selective up-regulation could thus be of therapeutic potential in colitis.

Ozonation of Human Blood Induces a Remarkable Upregulation of Heme Oxygenase-1 and Heat Stress Protein-70

Heme oxygenase-I (HO-1) has emerged as one of the most protective enzymes and its pleiotropic activities have been demonstrated in a variety of human pathologies. Unpublished observations have shown that HO-1 is induced after the infusion of ozonated blood into the respective donors, and many other experimental observations have demonstrated the efficacy of oxidizing agents. It appeared worthwhile to evaluate whether we could better define the activity of potential inducers such as hydrogen peroxide and ozonated human plasma. Human vascular endothelial cells at confluence were challenged with different concentrations of these inducers and the simultaneous production of nitric oxide (NO); and HO-1 was measured by either measuring nitrite, or bilirubin formation, or/and the immune reactivity of the protein by Western blot using a rabbit antihuman HO-1 and Hsp-70. The results show that production of both NO and HO-1 is fairly dose dependent but is particularly elevated using human plasma after transient exposure to a medium ozone concentration. At this concentration, there is also induction of Hsp-70. The results clarify another positive effect achievable by the use of ozone therapy.

The Frequency of UDP-Glucuronosyltransferase 1A1 Promoter Region (TA)7 Polymorphism in Newborns and it's Relation with Jaundice

Increased bilirubin formation and decreased bilirubin conjugation play an important role in the pathogenesis of the newborn jaundice. Although physiologic jaundice is seen in most of the newborns, there are many risk factors that affect the severity and duration of hyperbilirubinemia. The latest studies showed that the frequency and severity of neonatal jaundice have been increased when mutations of the gene coding UDP-glucuronosyltransferase(UGT)1A1 coexist with other risk factors. Healthy term newborns weighing over 2500 g. were included in this study. The patient group consisted of 107 newborns either with total bilirubin level over 15 mg dl(-1) within 7 days or 5 mg dl(-1) after 15 days of age. The control group consisted of 55 newborns with bilirubin levels in physiological ranges. We investigated the frequency of promoter region [thymine-adenine(TA)]7 polymorphism in UGT1A1 gene. Factors which might cause pathologic and prolonged jaundice with coexisting polymorphism were also investigated. UGT1A1 6/7 genotype was found to be 11% in patient group and 13% in the control group. The difference between patient and control groups was not statistically significant. (TA)7 allele frequency was 0.069 and it is concluded that UGT1A1 promoter region polymorphism was not a risk factor for neonatal jaundice.