Intraperitoneally administered biliverdin protects against UVB-induced skin photo-damage in hairless mice.

Oxidative stress is shown to be responsible for ultraviolet B (UVB) irradiation-induced skin cancer and premature aging. Biliverdin (BVD), a product of heme oxygenase-1, has strong anti-oxidant and anti-inflammatory properties. In the present study, we investigated the effects of BVD on UVB-induced skin photo-damage in hairless mice. Mice were divided into three groups: control group, UVB group (only UVB irradiation) and BVD+UVB group (mice were intraperitoneally injected with BVD before each UVB irradiation). Intraperitoneal BVD injection resulted in a significant photoprotective effect by reducing morphological and histopathological changes to the skin. BVD also exhibited a significant antioxidant effect by increasing the superoxide dismutase (SOD) level and decreasing the thiobarbituric acid reactive substances (TBARS) level compared with the control group. In addition, BVD activated biliverdin reductase (BVR) expression and inhibited the UVB-induced increase of p38 mitogen-activated protein kinase phosphorylation (p-p38MAPK), MMP (matrix metalloproteinase)-1 and MMP-3 expression (p<0.05). It also significantly decreased the interleukin (IL)-6 level compared with the UVB group (p<0.05). In conclusion, these data suggest that the intraperitoneally administered BVD can prevent UVB irradiation-induced skin photo-damage in hairless mice and that this is likely mediated by its antioxidant and anti-inflammatory mechanisms and cell signal regulatory action.

Biliverdin's regulation of reactive oxygen species signalling leads to potent inhibition of proliferative and angiogenic pathways in head and neck cancer.

BACKGROUND: In this study, we evaluate whether the use of biliverdin (BV), a natural non-toxic antioxidant product of haeme catabolism, can suppress head and neck squamous cell carcinoma (HNSCC) cell proliferation and improve the tumour survival both in vitro and in vivo. Furthermore, we investigate whether this therapeutic outcome relies on BV's potent antioxidant effect on reactive oxygen species (ROS)-mediated signalling. METHODS: Two well-characterised HNSCC cell lines and a mouse model with human HNSCC were used for this study. In vitro, the effect of BV on ROS was assayed. Subsequently, critical regulatory proteins involved in growth, antiapoptotic, and angiogenic pathways were investigated by western blot analysis. In addition, the antiproliferative effect of BV was also evaluated using the clonogenic assay. Moreover, tumour growth inhibition was assessed using a mouse model with HNSCC. RESULTS: Biliverdin treatment resulted in decreased ROS, leading to suppression of proliferation and angiogenesis pathways of HNSCC, significantly decreasing the expression and phosphorylation of oncogenic factors such as epidermal growth factor receptor (EGFR), phosphorylation of Akt, and expression of angiogenic marker and transcription factor, hypoxia-inducible factor1-α (HIF1-α). Furthermore, this downregulation of ROS by BV led to a significant suppression of tumour growth in vivo. CONCLUSIONS: Our study demonstrates the efficacy of a novel therapeutic approach using BV as an antitumour agent against HNSCC through its effect on EGFR/Akt and HIF1-α/angiogenesis signal transduction pathways. Our findings indicate that BV's inhibitory effect on these tumorigenic pathways relies on its antioxidant effect, and may extend its therapeutic potential to other solid cancers.

Effects of biliverdin administration on acute lung injury induced by hemorrhagic shock and resuscitation in rats.

Hemorrhagic shock and resuscitation induces pulmonary inflammation that leads to acute lung injury. Biliverdin, a metabolite of heme catabolism, has been shown to have potent cytoprotective, anti-inflammatory, and anti-oxidant effects. This study aimed to examine the effects of intravenous biliverdin administration on lung injury induced by hemorrhagic shock and resuscitation in rats. Biliverdin or vehicle was administered to the rats 1 h before sham or hemorrhagic shock-inducing surgery. The sham-operated rats underwent all surgical procedures except bleeding. To induce hemorrhagic shock, rats were bled to achieve a mean arterial pressure of 30 mmHg that was maintained for 60 min, followed by resuscitation with shed blood. Histopathological changes in the lungs were evaluated by histopathological scoring analysis. Inflammatory gene expression was determined by Northern blot analysis, and oxidative DNA damage was assessed by measuring 8-hydroxy-2' deoxyguanosine levels in the lungs. Hemorrhagic shock and resuscitation resulted in prominent histopathological damage, including congestion, edema, cellular infiltration, and hemorrhage. Biliverdin administration prior to hemorrhagic shock and resuscitation significantly ameliorated these lung injuries as judged by histopathological improvement. After hemorrhagic shock and resuscitation, inflammatory gene expression of tumor necrosis factor-α and inducible nitric oxide synthase were increased by 18- and 8-fold, respectively. Inflammatory gene expression significantly decreased when biliverdin was administered prior to hemorrhagic shock and resuscitation. Moreover, after hemorrhagic shock and resuscitation, lung 8-hydroxy-2' deoxyguanosine levels in mitochondrial DNA expressed in the pulmonary interstitium increased by 1.5-fold. Biliverdin administration prior to hemorrhagic shock and resuscitation decreased mitochondrial 8-hydroxy-2' deoxyguanosine levels to almost the same level as that in the control animals. We also confirmed that biliverdin administration after hemorrhagic shock and resuscitation had protective effects on lung injury. Our findings suggest that biliverdin has a protective role, at least in part, against hemorrhagic shock and resuscitation-induced lung injury through anti-inflammatory and anti-oxidant mechanisms.

Heme oxygenase 1-generated carbon monoxide and biliverdin attenuate the course of experimental necrotizing pancreatitis.

OBJECTIVE: The cytoprotective enzyme heme oxygenase 1 (HO-1) is highly up-regulated in acute pancreatitis (AP). In this study, we tested its metabolites as potential therapeutic agents for AP in rats. METHODS: Acute necrotizing pancreatitis was induced by retrograde intraductal injection of sodium taurocholate in rats. Biliverdin hydrochloride (BV HCl) (50 µmol/kg subcutaneously), the carbon monoxide, donor methylene chloride (MC) (500 mg/kg orally), or iron-chelating desferrioxamine (DFO) (125 mg/kg subcutaneously) were administered in a therapeutic manner starting with the first dose 4 hours after taurocholate injection to mimic the effects of HO-1 metabolites. RESULTS: Administration of BV HCl, MC, or DFO showed significant reduction of inflammatory activity in comparison to controls leading to lower myeloperoxidase activity in the pancreas, less edema, lower ascites volumes, and preservation of tissue integrity (P < 0.05). Administration of either BV HCl or MC markedly increased 5-day survival rate (70% and 75% vs 40%; P < 0.05), whereas DFO had no significant effect on survival (60%). When given in therapeutic manner, all 3 substances led to diminished nuclear factor κB activity in the pancreas (P < 0.05). CONCLUSIONS: Therapeutic use of BV HCl and MC led to marked reduction of mortality in experimental pancreatitis. Thus, HO-1 metabolites may present a novel therapeutic approach in AP treatment.

Heme oxygenase-1 end-products carbon monoxide and biliverdin ameliorate murine collagen induced arthritis.

OBJECTIVES: Heme oxygenase-1 (HO-1) which degrades Heme to free iron, biliverdin and carbon monoxide (CO) plays an important role in inflammation. There are, however, conflicting data concerning the role of HO-1 in rheumatoid arthritis (RA) and the therapeutic potential of individual heme degradation products remains to be determined. We therefore investigated the effect of CO and biliverdin upon therapeutic administration in the murine collagen induced arthritis (CIA) model of RA. METHODS: CIA was induced in DBA/1 mice. Anti-CII antibody levels were determined by ELISA. Mice were scored for paw swelling and grip strength. After the first clinical signs of arthritis one group of animals was treated with biliverdin, the second group was treated with CO. After 60 days all animals were sacrificed and analysed for histomorphological signs of arthritis. RESULTS: All animals immunised with CII developed serum anti-CII antibodies. Antibody levels were decreased in the CO-treated group. Both, Biliverdin and the CO-treated animals, showed an improvement in clinical disease activity. Histological analysis revealed significantly less inflammation, erosion and reduced numbers of osteoclasts in CO-treated animals only, whereas cartilage degradation was prevented in both biliverdin and CO-treated animals. CONCLUSIONS: Our data demonstrate a beneficial effect of CO, in particular, and biliverdin, on inflammation and bone destruction in the CIA mouse model.

Bile pigment pharmacokinetics and absorption in the rat: therapeutic potential for enteral administration.

BACKGROUND AND PURPOSE: Bilirubin and biliverdin possess antioxidant and anti-inflammatory properties and their exogenous administration protects against the effects of inflammation and trauma in experimental models. Despite the therapeutic potential of bile pigments, little is known about their in vivo parenteral or enteral absorption after exogenous administration. This study investigated the absorption and pharmacokinetics of bile pigments after i.v., i.p. and intraduodenal (i.d.) administration in addition to their metabolism and routes of excretion. EXPERIMENTAL APPROACH: Anaesthetized Wistar rats had their bile duct, jugular and portal veins cannulated. Bile pigments were infused and their circulating concentrations/biliary excretion were measured over 180 min. KEY RESULTS After i.v. administration of unconjugated bilirubin, biliverdin and bilirubin ditaurate, their plasma concentrations decreased exponentially over time. Subsequently, native and metabolized compounds appeared in the bile. When administered i.p., their absolute bioavailabilities equalled 14.0, 16.1 and 33.1%, respectively, and correspondingly 38, 28 and 34% of the same bile pigment doses were excreted in the bile. Administration of unconjugated bilirubin and bilirubin ditaurate i.d. increased their portal and systemic concentrations and their systemic bioavailability equalled 1.0 and 2.0%, respectively. Correspondingly, 2.7 and 4.6%, of the doses were excreted in the bile. Biliverdin was rapidly metabolized and these products were absorbed and excreted via the urine and bile. CONCLUSIONS AND IMPLICATIONS: Bile pigment absorption from the peritoneal and duodenal cavities demonstrate new routes of administration for the treatment of inflammatory and traumatic pathology. Oral biliverdin administration may lead to the production of active metabolite that protect from inflammation/complement activation.

Biliverdin Rescues the HO-2 Null Mouse Phenotype of Unresolved Chronic Inflammation Following Corneal Epithelial Injury.

PURPOSE. The heme oxygenase system (HO-1 and HO-2) represents an intrinsic cytoprotective and anti-inflammatory pathway based on its ability to modulate leukocyte migration and to inhibit the expression of inflammatory cytokines and proteins by its products biliverdin/bilirubin and carbon monoxide. Corneal injury in HO-2 null mice leads to impaired healing and chronic inflammatory complications, including ulceration and neovascularization. The authors examined whether topically administered biliverdin can counteract the effects of HO deficiency in a corneal epithelial injury model. METHODS. HO-2 null mice were treated with biliverdin 1 hour before epithelial injury and twice a day thereafter. Reepithelialization and neovascularization were assessed by fluorescein staining and vital microscopy, respectively, and were quantified by image analysis. Inflammation was quantified by histology and Gr-1-specific immunofluorescence, and oxidative stress was assessed by DHE fluorescence. RESULTS. Treatment with biliverdin accelerated wound closure, inhibited neovascularization and reduced epithelial defects. It also reduced inflammation, as evidenced by a reduction in the appearance of inflammatory cells and the expression levels of inflammatory and oxidant proteins, including KC and NOXs. CONCLUSIONS. The results clearly show that biliverdin, directly or through its metabolism to bilirubin by biliverdin reductase-the expression of which is increased after injury-rescues the aberrant inflammatory phenotype, further underscoring the importance of the HO system in the cornea for the execution of an ordered inflammatory and reparative response.

Protection against lung graft injury from brain-dead donors with carbon monoxide, biliverdin, or both.

BACKGROUND: The process of brain death can induce acute lung injury in donors and aggravate ischemia-reperfusion injury in grafts. Carbon monoxide (CO) and biliverdin (BV) have been shown to attenuate ischemia-reperfusion injury. We therefore examined if the administration of both CO and BV provide enhanced cytoprotection against lung graft injury from brain-dead (BD) rat donors. METHODS: Brain death was induced in all donors, after which they were observed for 1.5 hours and then underwent lung transplantation. The recipients were ventilated with 40% oxygen (control group), ventilated with 250 ppm CO in 40% oxygen (CO group), treated with BV (35 mg/kg) intraperitoneally (BV group), or treated with CO and BV conjointly (COBV group) before transplantation (n = 8 each group). The recipients were sacrificed 2 hours after lung transplantation by exsanguination. Serum levels of interleukin (IL)-8 and tumor necrosis factor (TNF)-α were measured by enzyme-linked immunosorbent assay. RESULTS: CO and/or BV treatment attenuated partial pressure of arterial oxygen (Pao(2))/fraction of inspired oxygen (Fio(2)) aggravation in the recipients after reperfusion, reduced the wet weight/dry weight ratio, decreased the lung injury score, inhibited the activity of myeloperoxidase in grafts, and decreased serum levels of IL-8 and TNF-α compared with the control group (p < 0.05). The COBV group had significantly decreased malonaldehyde levels and increased superoxide dismutase levels in lung grafts compared with the CO group (p < 0.05). The static pressure-volume curve of the lungs was ameliorated in the CO group, BV group, and COBV group compared with the control group (p < 0.05). CONCLUSIONS: CO and BV exert protective effects through anti-inflammatory and anti-oxidant mechanisms, and dual treatment provided enhanced cytoprotection against lung graft injury from BD rat donors.

Biliverdin-induced brainstem auditory evoked potential abnormalities in the jaundiced Gunn rat.

Brainstem auditory evoked potential (BAEP) abnormalities occur in jaundiced Gunn rats given sulfadimethoxine to displace bilirubin bound to serum albumin, releasing it into the tissues. One problem with the model is that after displacement, plasma bilirubin levels drop and do not correlate with neurological dysfunction. In this report, we administered biliverdin, the immediate precursor of bilirubin, in 15- to 17-day-old Gunn rat pups to create an improved model of bilirubin-induced neurological dysfunction. Total plasma bilirubin (TB) levels were measured with a Leica bilirubinometer. Biliverdin (40 mg/kg) or phosphate-buffered saline (PBS) was administered either once and BAEPs recorded 8 h later or twice, 12 h apart, and BAEPs recorded 24 h after the initial injection. A single biliverdin injection produced a significantly decreased amplitude of BAEP wave III, 1.21+/-0.25 vs. 0.49+/-0.27 microV (control vs. biliverdin). The two-injection paradigm resulted in a significantly elevated TB (9.9+/-1.2 vs. 14.9+/-3.1 mg/dl; control vs. biliverdin), significant increases in I-II (1.15+/-0.08 vs. 1.42+/-0.09 ms) and I-III (2.17+/-0.08 vs. 2.5+/-0.13 ms) interwave intervals and a decrease in the amplitude of wave III (1.36+/-0.30 vs. 0.38+/-0.26 microV). Additionally, there were significant correlations between TB and the amplitude of wave III (r2=0.74) and TB and the I-III interwave interval (r2=0.51). In summary, biliverdin administration in jaundiced Gunn rat pups produces BAEP abnormalities consistent with those observed in the sulfadimethoxine model and human newborn hyperbilirubinemia and resulted in increased plasma bilirubin levels that correlate with the degree of neurological dysfunction.

Biliverdin protects against polymicrobial sepsis by modulating inflammatory mediators.

Highly inducible heme oxygenase (HO)-1 is protective against acute and chronic inflammation. HO-1 generates carbon monoxide (CO), ferrous iron, and biliverdin. The aim of this study was to investigate the protective effects of biliverdin against sepsis-induced inflammation and intestinal dysmotility. Cecal ligation and puncture (CLP) was performed on Sprague-Dawley rats under isoflurane anesthesia with and without intraperitoneal biliverdin injections, which were done before, at the time of CLP, and after CLP. In vivo gastrointestinal transit was carried out with fluorescein-labeled dextran. Jejunal circular muscle contractility was quantified in vitro using organ bath-generated bethanechol dose-response curves. Neutrophilic infiltration into the muscularis externa was quantified. The jejunal muscularis was studied for cytokine mRNA expressions [interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, inducible nitric oxide synthase, cyclooxygenase-2, biliverdin, IL-10, and HO-1] using real-time RT-PCR. Biliverdin treatment prevented the sepsis-induced suppression of gastrointestinal muscle contractility in vivo and in vitro and significantly decreased neutrophilic infiltration into the jejunal muscularis. Inflammatory mRNA expressions for small bowel IL-6 and MCP-1 were significantly reduced after biliverdin treatment in CLP-induced septic animals compared with untreated septic animals. The anti-inflammatory mediator expression of small bowel IL-10 was significantly augmented after CLP at 3 h compared with untreated septic animals. These findings demonstrate that biliverdin attenuates sepsis-induced morbidity to the intestine by selectively modulating the inflammatory cascade and its subsequent sequelae on intestinal muscularis function.

Biliverdin administration protects against endotoxin-induced acute lung injury in rats.

Given the high morbidity and mortality rates associated with pulmonary inflammation in sepsis, there is a pressing need for new therapeutic modalities to prevent acute respiratory distress. The enzyme heme oxygenase-1 (HO-1) provides potent cytoprotection against lung injury; however, the mechanism by which it does so is unclear. HO-1 catabolizes heme into biliverdin (BV), which is rapidly converted to bilirubin by BV reductase. We tested the hypothesis that BV administration could substitute for the effects observed with HO-1. Using the well-described rat model of LPS-induced shock, we demonstrate that exposure to BV imparts a potent defense against lethal endotoxemia systemically, as well as in the lungs, and effectively abrogates the inflammatory response. BV administration before a lethal dose of LPS leads to a significant improvement in long-term survival: 87% vs. 20% in sham-treated controls. BV treatment suppressed LPS-induced increases in lung permeability and lung alveolitis and significantly reduced serum levels of the LPS-induced proinflammatory cytokine IL-6. Moreover, bilirubin administered just after LPS also abrogated lung inflammation. BV treatment also augmented expression of the anti-inflammatory cytokine IL-10. Similar effects on production were observed with BV treatment in vitro in mouse lung endothelial cells and RAW 264.7 macrophages treated with LPS. In conclusion, these data demonstrate that BV can modulate the inflammatory response and suppress pathophysiological changes in the lung and may therefore have therapeutic application in inflammatory disease states of the lung.

Carbon monoxide is the heme oxygenase product with a pyretic action: evidence for a cGMP signaling pathway.

We have recently reported that the central heme oxygenase (HO) pathway has an important role in the genesis of lipopolysaccharide fever. However, the HO product involved, i.e., biliverdine, free iron, or carbon monoxide (CO), has not yet been identified with certainty. Therefore, in the present study, we tested the thermoregulatory effects of all HO products. Body core temperature (T(c)) and gross activity of awake, freely moving rats was measured by biotelemetry. Intracerebroventricular administration of heme-lysinate (152 nmol), which induces the HO pathway, evoked a marked increase in T(c), a response that was attenuated by intracerebroventricular pretreatment with the HO inhibitor zinc deuteroporphyrin 2,4-bis glycol (200 nmol), indicating that an HO product has a pyretic action in the central nervous system (CNS) of rats. Besides, heme-lysinate also increased gross activity, but no correlation was found between this effect and the increase in T(c). Moreover, intracerebroventricular biliverdine or iron salts at 152 nmol, a dose at which heme-lysinate was effective in increasing T(c), produced no change in T(c). Accordingly, intracerebroventricular treatment with the iron chelator deferoxamine elicited no change in basal T(c) and did not affect heme-induced pyresis. However, heme-induced pyresis was completely prevented by the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxaline-1-one. Because biliverdine and iron had no thermoregulatory effects and CO produces most of its actions via sGC, these data strongly imply that CO is the only HO product with a pyretic action in the CNS.

A beneficial role of bile pigments as an endogenous tissue protector: anti-complement effects of biliverdin and conjugated bilirubin.

Bile pigments possess an anti-complement property and could be involved in tissue protection. In this study, we examined the physiological actions of bile pigments, which had been generally regarded as waste catabolites. Biliverdin inhibited complement cascade reactions in vitro, especially at the C1 step in the classical pathway at low micromolar concentrations. Further, Forssman anaphylaxis in guinea pigs, being closely associated with complement reactions, was inhibited by oral or intravenous administration of biliverdin. Conjugated bilirubin also showed an inhibitory effect on complement-dependent reactions in vitro. From these observations, we propose a hypothesis that the pigments serve as endogenous tissue protectors by multiple lines of mechanisms including antioxidant and anti-complement actions.

Inhibitory effect of porphyrins on the proliferation of mouse spleen lymphocytes in vitro.

The influence of various porphyrins (deuteroporphyrin IX, mesoporphyrin IX, protoporphyrin IX, hematoporphyrin) and two related compounds (hemin, biliverdin) on the spontaneous proliferation of mouse spleen lymphocytes has been estimated in vitro by the 3H-thymidine uptake assay. It has been found that porphyrins (endogenous ligands for the mitochondrial benzodiazepine receptor) produce a concentration-dependent inhibition of 3H-thymidine incorporation into the DNA of these cells. Metalloporphyrin-hemin has been observed to evoke a weak inhibitory effect, in a high concentration (10(-4)M), whereas biliverdin, a porphyrins degradation product, was inactive in the same experimental conditions. Those findings indicate that endogenous porphyrins, presumably acting through the mitochondrial benzodiazepine receptor, could regulate the proliferation of mouse spleen lymphocytes in vitro.

[Possible role of biliverdin as an initiator of liver regeneration]. / Posible papel de la biliverdina como iniciador de la regeneración hepática.

Biliverdin has been proposed as the biological signal that triggers liver regeneration after partial hepatectomy, since a transitory increase of plasma levels is found shortly after partial hepatectomy. We have carried out a detailed study to establish the feasibility of such a hypothesis. When biliverdin is administered i.p., it binds to albumin. This became evident by electrophoresis and affinity chromatography. Biliverdin disappears from the peritoneal cavity following a 1st order kinetics (t 1/2 = 40 min, K = 0.0175 min -1). Biliverdin reductase activity in the peritoneal exudate was high (1.04 mumol Bv min -1/mg protein), and very small amounts of biliverdin in comparison to the administered dose were found in plasma. However, unconjugated bilirubin level was high in plasma and the conjugated bilirubin in bile flux reached its maximum 90 min after administration. On the basis of blood elimination of biliverdin when administered intravenously (t 1/2 = 11 min, K = 0.068 min -1), a series of experiments was designed to reproduce the levels of plasma biliverdin as found shortly after partial hepatectomy. Our results showed a total absence of liver regeneration. There is, however, increase of the mitotic index if biliverdin is administered intraperitoneally. This effect is not directly related to biliverdin but to unspecific (?) stimulation of peritoneal cavity since the same effect was obtained after administration of unrelated substances (diatomaceous earth, killed bacteria). The mitotic figures did not incorporate 3H-thymidine, and it is suggested that those cells came from a G2 blocked quiescent cell population in liver. We therefore conclude that biliverdin is not the physiological trigger of liver regeneration.