Biochemical properties of the heme oxygenase inhibitor, Sn-protoporphyrin. Interactions with apomyoglobin and human serum albumin

Sn-protoporphyrin is a strong competitive inhibitor of heme oxygenase and a potential pharmacological agent for the treatment of neonatal hyperbilirubinemia. Little is otherwise known about the biochemistry of tin porphyrins. We have investigated aspects of the chemistry of tin-protoporphyrin in aqueous solution and of its interactions with heme-binding proteins other than heme oxygenase, specifically apomyoglobin and human serum albumin. In the pH region 7-10, Soret region absorption studies of unbound Sn-protoporphyrin demonstrate a pH-dependent monomer-dimer equilibrium (KD congruent to 10(6) M-1 at pH 7) with little higher aggregation. Dissociation of the dimer is relatively slow at neutral pH, permitting interaction of protein ligands with monomeric and dimeric species to be distinguished and providing insights into kinetic mechanisms of porphyrin binding by heme-binding proteins. In the present study, the kinetics of interaction of Sn-protoporphyrin with apomyoglobin are presented as novel evidence that this binding proceeds by an induced fit mechanism. Binding of Sn-protoporphyrin to both apomyoglobin and serum albumin is unexpectedly weak. Between pH 7 and 9, the apparent affinity of Sn-protoporphyrin for apomyoglobin is less than 1/200 that of heme and, at pH 9, is also significantly less than that of protoporphyrin. The apparent affinity of Sn-protoporphyrin for human serum albumin is less than 1/1000 that of heme and 1/30 to 1/100 that of protoporphyrin. Competition studies between heme and Sn-protoporphyrin and between bilirubin and Sn-protoporphyrin indicate that Sn-protoporphyrin distributes differently among porphyrin-binding sites on serum albumin than does heme and that it is also not an effective competitor with bilirubin for bilirubin-binding sites. These results argue that Sn-protoporphyrin should not significantly alter normal mechanisms for the binding and transport of heme or of preformed bilirubin by serum albumin. From a more general perspective, the results indicate potentially unusual binding site selectivity by tin chelates; possible origins of this selectivity are discussed.     

Fluorometric measurement of tin-protoporphyrin in biological samples

Sn-protoporphyrin is a potent competitive inhibitor of heme oxygenase, can suppress neonatal and other forms of hyperbilirubinemia in laboratory animals, and represents a potential new approach to the treatment of neonatal jaundice in humans. In order to study the disposition of Sn-protoporphyrin in vivo we have developed a sensitive fluorometric method for the quantitation of this metalloporphyrin in biological samples. The method is sensitive to concentrations as low as 0.01 nmol/ml, and is specific for Sn-protoporphyrin even in the presence of other porphyrins such as protoporphyrin.     

Protective effect of Sn-protoporphyrin against doxorubicin-induced perturbations of heme metabolism

The administration of doxorubicin, an anti-tumor antibiotic, to rodents resulted in an increase in heme oxygenase activity and a decrease in delta-aminolevulinate (ALA) synthase activity and in cellular heme and cytochrome P450 content in liver. Sn-protoporphyrin, a potent inhibitor of heme degradation both in vitro and in vivo, when administered to rodents prior to doxorubicin, mitigates the drug-induced toxic actions which are reflected by the drug-induced decreases of both cellular heme and cytochrome P450 content. Sn-protoporphyrin thus provides a pharmacological means of protecting against the toxic effects of doxorubicin and other drugs which enhance heme oxygenase activity and thus decrease cellular heme and cytochrome P450 content in vivo.     

HO-1 在肝肾疾病中的作用研究进展

血红素氧合酶是血红素降解的限速酶,与酶解产物胆红素、CO-道,共同发挥着抗氧化、抗炎、抑制细胞凋亡、改善组织微循环等作用。血红素氧合酶1是血红素氧合酶的诱导型在脓毒血症、高血压、急性肺损伤等多种疾病中均呈现适应性诱导表达并产生相应的细胞保护作用在肝脏缺血再灌注损伤、肝硬化、肝衰竭、肝移植、急性肾损伤、移植肾损伤等疾病中也发挥着细胞保护作用。本文综述了近年来血红素氧合酶1在肝肾疾病中作用的研究进展,以期为未来治疗方法带来新突破。     

The influence of side chain modifications of the heme moiety on prosthetic acceptance and function of rat hepatic cytochrome P-450 and tryptophan pyrrolase

The relative potential of various structural isomers (III, XIII) and various 2,4-side chain modified analogs of heme (iron-protoporphyrin IX) to incorporate into rat liver hemoproteins, cytochrome P-450(s), and tryptophan pyrrolase was examined. Such assessments for hepatic cytochrome P-450 relied on generation of reconstitutible apocytochrome(s) P-450 by suicidal alkylation of the existing prosthetic heme moiety by allylisopropylacetamide (AIA) in vivo. Subsequent replacement of the prosthetic heme was brought about by incubating the apocytochrome(s) P-450-enriched preparations with a particular heme isomer or analog. Structure-function relationships of the reconstituted isozymes were assessed in microsomal preparations by monitoring cytochrome P-450 content (structure) and its mixed function oxidase activity (function). In parallel, the relative ability of these heme isomers and analogs to functionally constitute hepatic tryptophan pyrrolase was also assessed by monitoring the relative increase in holoenzyme activity when preparations deliberately enriched in constitutible apoenzyme were incubated with each of these compounds. The findings reveal that 2,4-side chain modifications on the heme IX skeleton markedly influence the function of the constituted hemoproteins possibly by affecting their structural assembly through steric, electronic, and/or hydrophobic interactions with the corresponding apoproteins. Furthermore, these studies not only reveal that the structural specifications of the active prosthetic site of rat liver cytochrome P-450(s) differ from those of tryptophan pyrrolase, but also that the structural specifications of these mammalian hemoproteins for their prosthetic heme differ considerably from those reported for their bacterial counterparts.     

Bilirubin is highly effective in preventing in vivo delta-aminolevulinic acid-induced oxidative cell damage

Delta-aminolevulinic acid (ALA), precursor of heme, accumulates in a number of organs, particularly in liver of patients with acute porphyrias or lead intoxication. This study characterizes the involvement of bilirubin as an antioxidant in a chronic intoxication with ALA. Female Wistar rats were injected intraperitoneally a daily dose of 40 mg ALA/body wt., during 10 days. A marked increase in lipid peroxidation and a decrease in GSH content were observed 24 h after the last injection of ALA. The activities of liver antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase were also diminished. ALA synthase (ALA-S) and heme oxygenase-1 were induced. Both ALA dehydratase (ALA-D) and porphobilinogenase (PBG-ase) activities were inhibited. Administration of bilirubin (5 mmol/kg body wt.) 2 h before ALA treatment entirely prevented the effects of ALA. Co-administration of ALA and Sn-protoporphyrin IX (Sn-PPIX; 100 microg/body wt., i.p.), a potent inhibitor of heme oxygenase, completely abolished its induction and provoked a marked decrease in liver GSH levels as well as an increase in lipid peroxidation. These results add further support to the proposal assigning bilirubin a key protective role against oxidative damage here induced by ALA.     

Heme catabolism in cultured hepatocytes: evidence that heme oxygenase is the predominant pathway and that a proportion of synthesized heme is converted rapidly to biliverdin

Heme oxygenase has been considered to be involved in the predominant pathway of heme degradation in vivo. However, alternative pathways involving cytochrome P-450 reductase, and lipid peroxidation, have previously been demonstrated in vitro, and studies with cultured rat hepatocytes were interpreted to show a majority of endogenous hepatic heme breakdown by non-heme oxygenase pathways. To clarify the pathway of heme breakdown in hepatocytes and the role of heme oxygenase in this process, cultured hepatocytes were pre-labelled with 5-[5-14C]aminolevulinate [( 14C]ALA). Radioactivity in heme, carbon monoxide, and bile pigments was measured for 8-24 h after the removal of [14C]ALA. In cultured chick embryo hepatocytes, which lack biliverdin reductase, the rate of production of biliverdin IXa was closely similar to the rate of catabolism of exogenous heme and radioactivity in carbon monoxide and biliverdin IXa was similar to the loss of radioactivity from endogenous heme. These results support the conclusion that heme breakdown occurred predominantly, if not solely, by heme oxygenase. Also, no evidence of non-heme oxygenase pathways was found in the presence of tin protoporphyrin, an inhibitor of heme oxygenase or mephenytoin, an inducer of both cytochrome P-450 and heme oxygenase. Similarly, in untreated cultured rat hepatocytes, radioactivity in carbon monoxide corresponded with loss of radioactivity in endogenous heme. In other experiments with chick hepatocyte cultures, rates of heme synthesis and breakdown were measured, and data were fitted to various models of hepatic heme metabolism. The results observed were consistent only with models in which an appreciable fraction (control cells, 17%, mephenytoin treated cells, 41%) of the newly synthesized heme was degraded rapidly to biliverdin.     

Tin-protoporphyrin inhibits heme oxygenase and prevents the decline in hepatic heme and cytochrome P-450 contents produced in nude mice by tumor transplantation

Heme and hemeprotein perturbations are present in nude mice bearing transplanted tumors. Hepatic microsomal heme oxygenase activity is increased 50-100% in tumor bearing nu/nu mice when compared with normal controls. This elevation in activity of the rate-limiting enzyme of heme degradation is associated with a 50% depletion of microsomal heme and cytochrome P-45 concentrations in liver. The synthetic heme analogue, Sn-protoporphyrin, a potent inhibitor of heme oxygenase, lowers the activity of heme oxygenase in tumor bearing animals to below control levels. This effect is associated with a normalization of hepatic heme and cytochrome P-450 contents. These findings might have implications for protecting normal cells during tumor growth and chemotherapy.     

Mechanism of action of heme oxygenase. A study of heme degradation to bile pigment by 18O labeling

The formation of bile pigment from heme by a reconstituted heme oxygenase system containing purified bovine spleen heme oxygenase, NADPH-cytochrome P-450 reductase, and biliverdin reductase was studied under an atmosphere containing 18,18O2. The product, bilirubin, was isolated and subjected to mass spectrometry, which revealed incorporation of 18O consistent with a two-molecule mechanism, whereby the product bile pigment contains oxygen atoms derived from two different oxygen molecules.     

Bilirubin and ferritin as protectors against hemin-induced oxidative stress in rat liver.

The in vivo effect of hemin on both hepatic oxidative stress and heme oxygenase induction was studied. A marked increase in lipid peroxidation was observed 1 hr after hemin administration. Heme oxygenase-1 activity and expression appeared 6 hr after treatment, reaching a maximum between 12 and 15 hr after hemin administration. Such induction was preceded by a decrease in the soluble and enzymatic defenses, both effects taking place some hours before induction of heme oxygenase. Ferritin content began to increase 6 hr after heme oxygenase induction, and these increases were significantly higher 15 hr after treatment and remained high for at least 24 hr after hemin injection. Co-administration of tin protoporphyrin IX, a potent inhibitor of heme oxygenase, completely prevented the enzyme induction and the increase in ferritin levels, increasing the appearance of oxidative stress parameters. Administration of bilirubin, prevented the heme oxygenase induction as well as the decrease in hepatic GSH and the increase of lipid peroxidation when it was administered 2 hr before hemin treatment. These results indicate that the induction of heme oxygenase by hemin may be a general response to oxidant stress, by increasing bilirubin and ferritin levels and could therefore provide a major cellular defense mechanism against oxidative damage.     

Cadmium-mediated inhibition of testicular heme oxygenase activity: the role of NADPH-cytochrome c (P-450) reductase

The concerted activity of two microsomal enzymes, heme oxygenase and NADPH-cytochrome c (P-450) reductase, is required for isomer-specific oxidation of heme molecule; heme oxygenase is commonly believed to be rate limiting in this activity. In this report, we provide evidence strongly suggesting the rate-limiting role of the reductase in oxidation of heme molecule in rat testis. In the testis and the liver of rats treated with Cd (20 mumol/kg, sc, 24 h) heme oxygenase activity, assessed by the formation of bilirubin, was decreased by 50% and increased by 7-fold, respectively. In these animals, the reductase activity was decreased by nearly 75% in the testis, but remained unchanged in the liver. Similarly, the reductase activity in the liver was not altered when heme oxygenase activity was increased by 20-fold in response to bromobenzene treatment. Addition of purified testicular reductase preparation (purified over 4000-fold), or hepatic reductase, to the testicular microsomes of Cd-treated rats obliterated the Cd-mediated inhibition of heme oxygenase activity. The chromatographic separation of heme oxygenase and the reductase of the testicular microsomal fractions revealed that the reductase activity was markedly decreased (75%) while the heme oxygenase activity, when assessed in the presence of exogenous reductase, was not affected by in vivo Cd treatment. In vitro, the membrane-bound reductase preparation obtained from the testis was more sensitive to the inhibitory effect of Cd than the liver preparation. However, the purified reductase preparations from the testis and the liver exhibited a similar degree of sensitivity to Cd. Based on the molar ratio of heme oxygenase to the reductase in the microsomal membranes of the liver and the testis it appeared that the testicular heme oxygenase, which is predominantly HO-2 isoform, interacts with the reductase less effectively than HO-1; in the induced liver, heme oxygenase is predominantly the HO-1 isoform. It is suggested that due to the low abundance of NADPH-cytochrome c (P-450) reductase and the apparently lower affinity of the enzyme for HO-2, the reductase exerts a regulatory action on heme oxygenase activity in the testis.     

Regulation of rat hepatic delta-aminolevulinic acid synthetase and heme oxygenase activities: evidence for control by heme and against mediation by prosthetic iron

1. The effect of in vivo administration of 6 compounds on the activity of delta-aminolevulinic acid (ALA) synthetase and heme oxygenase were determined. 2. The order of decreasing potency in reducing ALA synthetase activity was heme, bilirubin, protoporphyrin IX, bilirubin dimethyl ester, CoCl2 and FeCl3. 3. The chelating agents EDTA and deferoxamine did not prevent heme's repression of ALA synthetase or induction of heme oxygenase activity. 4. The dose response, time course, enzyme subcellular distribution and chelation antagonism studies all suggest that heme itself, and not iron, regulates the rate limiting enzymatic steps of rat hepatic heme synthesis and degradation.     

Heme oxygenase induction by menadione bisulfite adduct-generated oxidative stress in rat liver

The in vivo effect of menadione bisulfite adduct on both hepatic oxidative stress and heme oxygenase induction was studied. A marked increase in lipid peroxidation was observed 1 h after menadione bisulfite adduct administration. To evaluate liver antioxidant enzymatic defenses, superoxide dismutase, catalase and glutathione peroxidase activities were determined. Antioxidant enzymes significantly decreased 3 h after menadione bisulfite adduct injection. Heme oxygenase activity appeared 6 h after treatment, peaking 9 h after menadione bisulfite adduct administration. Such induction was preceded by a decrease in the intrahepatic GSH pool and an increase in hydrogen peroxide steady-state concentration, both effects taking place some hours before induction of heme oxygenase. Iron ferritin levels and ferritin content began to increase 6 h after heme oxygenase induction, and these increases were significantly higher 15 h after treatment and remained high for at least 24 h after menadione bisulfite adduct injection. Administration of bilirubin entirely prevented heme oxygenase induction as well as the decrease in hepatic GSH and the increase in lipid peroxidation when administered 2 h before menadione bisulfite adduct treatment. These results indicate that the induction of heme oxygenase by menadione bisulfite adduct may be a general response to oxidant stress, by increasing bilirubin and ferritin levels and could therefore provide a major cellular defense mechanism against oxidative damage.     

Retinoic acid can enhance the stimulation by thyroid hormone of heme oxygenase activity in the liver of thyroidectomized rats.

The effects of retinoic acid (RA) (50 micrograms/100 g body wt. per day) on hepatic heme oxygenase activity, delta-aminolevulinate synthase (ALAS) activity and on cytochrome P-450 content were determined in thyroidectomized rats treated with T3 (10 micrograms/100 g body wt. per day) or diluent. RA, when administered for 3 days, failed to influence significantly the activity of either heme oxygenase or ALAS, however, the retinoid depleted hepatic cytochrome P-450 content by 17% (P less than 0.01) and microsomal heme content by 47% (P less than 0.001). T3 administration enhanced heme oxygenase activity by 72% (P less than 0.001) and ALAS activity by 251% (P less than 0.001) above levels in diluent treated controls and depleted cytochrome P-450 levels by 55% (P less than 0.001) and heme levels by 75% (P less than 0.001). When RA and T3 were administered together, the retinoid markedly enhanced the T3 stimulation of heme oxygenase activity; 173% above controls (P less than 0.001), and 61% above T3 alone (P less than 0.001). However, RA failed to influence the effect of T3 on ALAS activity or cytochrome P-450 depletion. The results indicate that RA can influence the levels of hepatic cytochrome P-450 and can modulate the stimulation of heme oxygenase activity by thyroid hormone in vivo.     

Inhibition of the biosynthesis of uroporphyrinogen and heme in rat liver during obstructive jaundice produced by bile duct ligation

Altered hepatic microsomal drug metabolism has been reported to occur in afflicted with hyperbilirubinemia. Similarities of the chemical structures of hydroxymethylbilane, an intermediate in the biosynthesis of uroporphyrinogen, to bilirubin prompted investigations of the effect of bilirubin on the activity of uroporphyrinogen I synthase (porphobilinogen deaminase, EC 4.3.1.8) and the biosynthesis of heme. Bilirubin was found to be a reversible, noncompetitive inhibitor of uroporphyrinogen I synthase. The inhibition constant (Ki) for bilirubin was 1.5 microM. Bile acids had no effect on rat hepatic uroporphyrinogen I synthase activity. Hyperbilirubinemia was achieved in rats by biliary ligation in order to investigate whether elevated levels of bilirubin impair the biosynthesis of hepatic heme in vivo. The relative rate of heme biosynthesis, as measured by the rate of incorporation of delta-[4-14C]aminolevulinic acid into heme, was decreased 59% 24 h after biliary obstruction. The levels of hepatic microsomal heme and cytochrome P-450 were decreased by 43 and 40%, respectively, 72 h after biliary obstruction. The activities of hepatic delta-aminolevulinic acid synthase and uroporphyrinogen I synthase were increased by 39 and 46%, respectively, 72 h after biliary obstruction. During the 48- to 72-h period following biliary obstruction, the urinary excretion of porphobilinogen and uroporphyrin was increased 3.0- and 3.5-fold, respectively, whereas, the urinary excretion of delta-aminolevulinic acid was not altered. During this 48-to 72-h time interval following biliary obstruction, 100% of the uroporphyrin was excreted as isomer I. These results indicate that bilirubin is capable of depressing the biosynthesis of rat hepatic heme and thus cytochrome P-450-mediated drug metabolism by inhibition of the formation of uroporphyrinogen. These findings are a plausible mechanism for reports of impaired clearance of various drugs in patients afflicted with hyperbilirubinemic disease states.     

Rat liver cytochrome P-450b, P-420b, and P-420c are degraded to biliverdin by heme oxygenase

In this report we provide data, for the first time, demonstrating the conversion of the heme moiety of certain cytochrome P-450 and P-420 preparations, to biliverdin, catalyzed by heme oxygenase. We have used purified preparations of cytochromes P-450c, P-450b, P-450/P-420c, or P-450/P-420b as substrates in a heme oxygenase assay system reconstituted with heme oxygenase isoforms, HO-2 or HO-1, NADPH-cytochrome c (P-450) reductase, biliverdin reductase, NADPH, and Emulgen 911. With cytochrome P-450b or P-450/P-420b preparations, a near quantitative conversion of degraded heme to bile pigments was observed. In the case of cytochrome P-450/P-420c approximately 70% of the degraded heme was accounted for as bilirubin but only cytochrome P-420c was appreciably degraded. The role of heme oxygenase in this reaction was supported by the following observations: (i) bilirubin formation was not observed when heme oxygenase was omitted from the assay system; (ii) the rate of degradation of the heme moiety was at least threefold greater with heme oxygenase and NADPH-cytochrome c (P-450) reductase than that observed with reductase alone; and (iii) the presence of Zn- or Sn-protoporphyrins (2 microM), known competitive inhibitors of heme oxygenase, resulted in 70-90% inhibition of bilirubin formation.     

Sodium arsenite induced alterations in bilirubin excretion and heme metabolism

The acute administration of sodium arsenite (AsIII) to rats resulted in a biphasic alteration of the hepatic cytosolic "free" heme pool. The first stage was an increase in the cytosolic "free" heme without significant effects on the content of cytochrome P-450 or on bilirubin excretion. The second stage consisted of a continuous fall of the cytosolic "free" heme and of the content of cytochrome P-450. These changes were concurrent with an eight-fold increase in heme oxygenase activity and associated with marked elevations in the biliary excretion of bilirubin. The bile was collected from chronically cannulated rats to avoid artifacts related to anesthesia or post anesthetic effects. The rapid increase in biliary excretion of labeled heme degradation products indicated an increased breakdown of newly synthesized heme. Immunoelectrophoresis of bile proteins showed an altered pattern of bile protein excretion. The increased biliary haptoglobin suggested some hemolysis, while the reduction in the free immunoglobulin A (IgA) secretory component showed an AsIII-related decreased protein transport across hepatocytes to bile. Further research is required to assess the direct role of an increased heme degradation in the genesis of the hepatotoxic effects of AsIII.     

Role of heme oxygenase-1 in hypoxia-reoxygenation: requirement of substrate heme to promote cardioprotection

Heme oxygenase-1 (HO-1) catalyzes the enzymatic degradation of heme to carbon monoxide, bilirubin, and iron. All three products possess biological functions; bilirubin, in particular, is a potent free radical scavenger of which its antioxidant property is enhanced at low oxygen tension. Here, we investigated the effect of severe hypoxia and reoxygenation on HO-1 expression in cardiomyocytes and determined whether HO-1 and its product, bilirubin, have a protective role against reoxygenation damage. Hypoxia caused a time-dependent increase in both HO-1 expression and heme oxygenase activity, which gradually declined during reoxygenation. Reoxygenation of hypoxic cardiomyocytes produced marked injury; however, incubation with hemin or bilirubin during hypoxia considerably reduced the damage at reoxygenation. The protective effect of hemin is attributable to increased availability of substrate for heme oxygenase activity, because hypoxic cardiomyocytes generated very little bilirubin when incubated with medium alone but produced substantial bile pigment in the presence of hemin. Interestingly, incubation with hemin also maintained high heme oxygenase activity levels during the reoxygenation period. Reactive oxygen species generation was enhanced after hypoxia, and hemin and bilirubin were capable once again to attenuate this effect. These results indicate that the HO-1-bilirubin pathway can effectively defend hypoxic cardiomyocytes against reoxygenation injury and highlight the issue of heme availability in the cytoprotective action afforded by HO-1.     

Targeted suppression of heme oxygenase-1 by small interference RNAs inhibits the production of bilirubin in neonatal rat with hyperbilirubinemia

Background  

Excessive accumulation of bilirubin contributes to neonatal hyperbilirubinemia in rats. Heme oxygenase (HO) is one of the rate-limiting enzymes in catabolizing heme to bilirubin. In the present study, we investigated whether suppression of rat HO-1 (rHO-1) expression by small interference RNAs (siRNAs) reduces bilirubin levels in hyperbilirubinemic rats.