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.     

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.     

Paraquat-generated oxidative stress in rat liver induces heme oxygenase-1 and aminolevulinic acid synthase

The in vivo effect of the known herbicide, paraquat, on both hepatic oxidative stress and heme metabolism was studied. A marked increase in lipid peroxidation and a decrease in reduced glutathione (GSH) content were observed 1 h after paraquat administration. The activity of liver antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase was decreased 3 h after paraquat injection. Heme oxygenase-1 induction started 9 h after treatment, peaking at 15 h. delta-aminolevulinic acid synthase induction occurred once heme oxygenase had been enhanced, reaching its maximum (1.5-fold of control) at 16 h. delta-aminolevulinic acid dehydratase activity was 40% inhibited at 3 h showing a profile similar to that of GSH, while porphobilinogenase activity was not modified along the whole period of the assay. Administration of alpha-tocopherol (35 mmol/kg body weight) 2 h before paraquat treatment entirely prevented the increase in thiobarbituric acid reactive substances (TBARS) content, the decrease in GSH levels as well as heme oxygenase-1 and delta-aminolevulinic acid synthase induction. This study shows that oxidative stress produced by paraquat leads to an increase in delta-aminolevulinic acid synthase and heme oxygenase-1 activities, indicating that the herbicide affects both heme biosynthesis and degradation.     

Heme regulation of HeLa cell transferrin receptor number

The number of diferic transferrin receptors on HeLa cells decreases when cells are grown in iron-supplemented media. The experiments reported here suggest that heme is the iron-containing compound which serves as the signal for receptor number regulation. When HeLa cells were grown in the presence of hemin, transferrin receptor number decreased to a greater degree than when cells were grown in equivalent amounts of iron supplied as ferric ammonium citrate. Incubation of cells in conditions which increased cellular heme content resulted in a decrease in cellular transferrin receptors. Incubating cells with 5-aminolevulinic acid (thus bypassing the rate-limiting step in heme biosynthesis, 5-aminolevulinic acid synthase) led to a decrease in transferrin receptor number. Incubation of cells with an inhibitor of heme oxygenase, Sn-protoporphyrin IX, also led to a decrease in transferrin receptor number. When cellular heme content was decreased by inhibiting heme synthesis with succinylacetone (an inhibitor of 5-aminolevulinic acid dehydratase), or by depriving cells of iron with deferoxamine, an increase in HeLa cell transferrin receptor number was seen. When HeLa cells were incubated with inducers of heme oxygenase (CoCl2, SnCl2, Co-protoporphyrin IX), transferrin receptor number also increased. The effects of all compounds which alter transferrin receptor number were dependent on the concentration of the supplement, as well as the duration of the supplementation. These experiments suggest that intracellular heme content may be an important signal controlling transferrin receptor number.     

Heme oxygenase activity in rat organs during cadmium chloride administration

Heme oxygenase activity, the level of spontaneous and ascorbat-induced LPO in the liver, kidney and spleen homogenates of rats and blood serum absorption spectrum in the Soret region in different periods both after CdCl2 and prior alpha-tocopherol administration were studied. The increase in the hemolysis products content in the serum was observed in 15 min after CdCl2 injection and remained during 24 h. Heme oxygenase activity in the liver and kidney increased after 6 h and stayed at the same level 24 h after CdCl2 administration. The level of spontaneous LPO in the spleen increased after 6 h, and in the liver and kidney the level of spontaneous and ascorbat-induced LPO increased in 24 h after CdCl2 injection. The preliminary alpha-tocopherol administration did not prevent the accumulation of hemolysis products in the serum and the increase of heme oxygenase activity in the liver and kidney caused by CdCl2 administration. However, the increase in the ascorbat-induced LPO in these organs was completely blocked. The role of heme and LPO in the heme oxygenase induction by CdCl2 are discussed.     

Regulation of delta-aminolevulinate synthase activity during the development of oxidative stress.

Activities of rat liver delta-aminolevulinate synthetase (delta-ALAS), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH), GSH content in the liver, and the absorption spectrum of blood serum were investigated after CoCl2, HgCl2, or beta-adrenoblocker (propranolol) injection and after CoCl2 and propranolol co-administration. Inhibition of the activity of the key heme biosynthesis enzyme delta-ALAS was most pronounced and prolonged during the first hours after CoCl2 and CoCl2 plus propranolol injections; this was associated with accumulation of Co2+--protoporphyrin-containing products of hemolysis. Inhibition of delta-ALAS after propranolol injection is not mediated by hemolysis. A decrease in GSH content precedes the induction of heme biosynthesis only in the case of HgCl2 administration, and this was associated with inhibition of GR and G6PDH. The decreased GSH content during the first hours after injection of propranolol and co-administration of CoCl2 and propranolol was not followed by increase in delta-ALAS activity 24 h after the injection. The mechanisms of the increase in the free heme content in the liver during the early stages of oxidative stress and the regulation of the key heme biosynthesis enzyme are discussed.     

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.     

Synergistic induction of delta-aminolevulinate synthase by glutethimide and iron: relationship to the synergistic induction of heme oxygenase

Relationships between activities of delta-aminolevulinate synthase and heme oxygenase, respectively the rate-limiting enzymes of heme biosynthesis and degradation, have been studied in chick embryo liver cell cultures following exposure of the cultures to glutethimide and iron, a combination known to produce a synergistic induction of both enzymes. In time-course experiments, synergistic induction of heme oxygenase activity by glutethimide and iron preceded that of delta-aminolevulinate synthase by 4 h. Effects of selective inhibitors of both heme synthesis and degradation have also been studied with respect to effects on delta-aminolevulinate synthase and heme oxygenase activities. The synergistic induction of heme oxygenase by glutethimide and iron appears to be dependent upon cellular heme synthesis because addition of inhibitors of heme biosynthesis, 4,6-dioxoheptanoic acid or N-methyl-mesoporphyrin abolishes this synergistic induction. Exposure of cultures to tin-mesoporphyrin, a potent inhibitor of heme oxygenase, prevented the synergistic induction of delta-aminolevulinate synthase produced by glutethimide and iron, or, when added after induction was already established, promptly halted any further induction. These results suggest that the level of activity of heme oxygenase can reciprocally modulate intracellular heme levels and thus activity of delta-aminolevulinate synthase.     

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.     

Involvement of heme oxygenase as antioxidant defense in soybean nodules

Objective: We have previously demonstrated that the inducible form of heme oxygenase plays a critical role in protecting against oxidative stress in mammals. To gain further insight into the functions of this enzyme in plants, we have tested its activity and expression in soybean nodules subjected to cadmium (Cd) stress.

Materials and methods: Four-weeks-old soybean nodulated plants were treated with different cadmium chloride concentrations (0, 50 and 200 μM) during 48 h. Oxidative stress parameters such as TBARS content, GSH levels and antioxidant enzyme activities were measured as well as heme oxygenase activity and expression. Besides, the effect of biliverdin and Zn-protophorphyrin IX were analized.

Results: Treatment with 200 μM Cd during 48 h caused a 67% increase in TBARS content, whereas GSH decreased 44%, and total superoxide dismutase, gluthatione reductase and guaiacol peroxidase were also inhibited 54, 20 and 60%, respectively. A total of 200 μM Cd produced the overexpression of heme oxygenase-1, as well as a 10-fold enhancement of its activity. Co-administration of biliverdin (10 μM) completely prevented the effects caused by Cd. Treatment with Zn protoporphyrin IX, a strong inhibitor of heme oxygenase, expectedly decreased heme oxygenase-1 activity to half. When the inhibitor was given together with Cd, completely prevented the enzyme induction and oxidative stress parameters were significantly enhanced.

Conclusion: Taking together, these results are indicating that heme oxygenase plays a protective role against oxidative cell damage in soybean nodules.     

Metabolism of heme and hemoproteins in rat liver upon administration of mercuric chloride

Rat liver delta-aminolevulinate synthase (delta-ALAS) activity in the early period after mercury chloride administration (0.7 mg per 100 g body weight) was found to be followed by free heme level increase, which was registered by the increase of heme saturation of the heme-binding protein tryptophan-2,3-dioxygenase (T-2,3-DO). delta-ALAS and heme oxygenase activity increase was observed 24 h after action. Microsomal cytochromes P450 and b5 levels decrease. Heme saturation of the T-2,3-DO returned to control level. Heme oxygenase and T-2,3-DO induction promoted hepatocytes free heme level normalization. Heme oxygenase and delta-ALAS induction role in the liver cells defense from the oxidative damage is discussed.     

Heme oxygenase exerts a protective role against oxidative stress in soybean leaves

We have previously demonstrated that the induction of heme oxygenase-1 (EC 1.14.99.3) plays a protective role for mammalian cells against oxidative stress. Here, we investigated for the first time the possible role of heme oxygenase-1 as an antioxidant defense in leaves of soybean plants. Treatment with 200 microM Cd during 48 h caused a 70% increase in thiobarbituric acid reactive substances, whereas GSH decreased 67%, guaiacol peroxidase and total superoxide dismutase also inhibited 49% and 46%, respectively. Two hundred micromolar of Cd produced the overexpression of heme oxygenase-1, as well as a 4.5-fold enhancement of its activity. Administration of biliverdin partially prevented the effects caused by Cd. Pretreatment with Zn protoporphyrin IX, a potent inhibitor of heme oxygenase, expectedly decreased heme oxygenase-1 activity to half. When the inhibitor was given before Cd, it completely prevented the enzyme induction increasing the levels of oxidative stress parameters. Collectively, these results indicated that although plant heme oxygenases share little homology to heme oxygenases from non-plant species, they also play an important protective role against oxidative cell damage.     

Activity of 5-aminolevulinate synthase in rat liver during degradation of cytochrome P-450 caused by administration of cadmium chloride

The 5-aminolevulinate synthase, tryptophan-2,3-dioxygenase activities and cytochrome P-450 content in the rat liver was studied in different terms after CdCl2 administration and after administration of metal salt against a background of 2-hours action of alpha-tocopherol. The lowering of activity of 5-aminolevulinate synthase in 2 h with the consequent increase of the enzyme activity in 6 h and 24 h was detected. The holoenzyme activity and heme saturation of tryptophan-2,3-dioxygenase increased 6 h after CdCl2 administration. The holoenzyme activity and the total activity of tryptophan-2,3-dioxygenase rised in 24 h. The level of cytochrome P-450 lowered. Preliminary administration of alpha-tocopherol prevented changes of studied parameters 24 h after CdCl2 administration. The relationship between decrease of cytochrome P-450 level and 5-aminolevulinate synthase activation are discussed.     

Induction of heme oxygenase mRNA by cobalt protoporphyrin in rat liver

The effect of cobaltic(III)-protoporphyrin on heme oxygenase activity and mRNA content was examined in vivo in the adult male rat liver. The activity of heme oxygenase, the rate-limiting enzyme in the degradation of heme, was enhanced, as expected, by cobalt protoporphyrin (25 mumol/kg body weight) in a time-dependent manner. Levels of enzyme activity were increased 2-fold by 8-16 h following treatment and were 6-fold higher than baseline values by 48 h. Administration of cobalt protoporphyrin resulted in a marked increase in heme oxygenase mRNA in the liver. Within 2 h of treatment, mRNA levels had increased 7.9-fold. The induction of heme oxygenase mRNA was maximal at 8 h when the levels were 58.5-fold above baseline. At every time point tested, the increase in heme oxygenase mRNA was several fold greater than that of enzyme activity.     

Properties of human kidney heme oxygenase: inhibition by synthetic heme analogues and metalloporphyrins

Heme oxygenase activities in human kidney microsomes were found to be from 0.238 to 0.620 nmol of bilirubin/mg/hr (mean 0.375, SD 0.134), which represent approximately 30% of activities determined for human adult liver. There was interindividual variation in heme oxygenase activity of a 2-5-fold difference. Rabbits were immunized with purified human liver heme oxygenase and the resulting antibody preparation was used to examine the species specificity of the enzyme. Microsomal protein with a molecular weight of 32,000 from human kidney was identified on Western blots by its reaction with the anti-heme oxygenase liver antibody similar to the purified enzyme protein. Thus, a homology exists between human hepatic and kidney heme oxygenase. The enzyme activity was sensitive to inhibition by metalloporphyrins, such as tin-protoporphyrin IX and, to a lesser degree, by zinc and cobalt protoporphyrin IX. In a study of different synthetic heme analogues for in vitro inhibition of heme oxygenase, we found that replacement of iron by zinc in deuteroporphyrin IX 2,4 bis glycol dramatically potentiated the inhibition of heme oxygenase activity. This finding demonstrated that zinc deuteroporphyrin IX 2,4 bis glycol is a most potent inhibitor of heme oxygenase activity.     

Induction of hepatic heme oxygenase activity by bromobenzene

Hepatic heme oxygenase, an enzyme which converts heme to carbon monoxide and bile pigment in vitro, is inducible by heme but also by large “toxic” doses of such nonheme substances as hormones, endotoxin, and heavy metal ions. When we gave rats a single hepatotoxic dose of allyl alcohol, ethionine, acetaminophen, furosemide, or endotoxin, hepatic heme oxygenase activity rose modestly (two- to fivefold) after 20 h. In contrast, administration of bromobenzene (5 mmol/kg) induced heme oxygenase in the liver an average of 15-fold after 20 h but was without effect on the enzyme in the kidney or spleen. The change in heme oxygenase was accompanied by a loss in cytochrome P-450 concentration and, in rats labeled with 5-δ-amino[¹⁴C]levulinic acid, an increased rate of degradation of hepatic [¹⁴C]heme to ¹⁴CO. Induction of heme oxygenase by bromobenzene was blocked by cycloheximide, an inhibitor of protein synthesis, but not by actinomycin D, an inhibitor of RNA synthesis. This suggests that bromobenzene stimulates de novo enzyme synthesis at the step of translation. Subtoxic doses of bromobenzene (less than 1 mmol/kg) gave proportionately greater induction of heme oxygenase. Furthermore, induction of the enzyme remained unaffected when bromobenzene hepatotoxicity was blocked by pretreatment of rats with SKF-525A, 3-methylcholanthrene, or cysteine (which supplements liver sulfhydryl content), or when hepatotoxicity was enhanced by pretreatment with phenobarbital or with diethylmaleate (which depletes hepatic glutathione). These data suggest that with induction of heme oxygenase by bromobenzene, neither liver cell necrosis nor alteration in hepatic sulfhydryl metabolism is indispensible. The latter characteristic differs from induction of the enzyme by metal ions in which depletion of sulfhydryl-containing constituents has been thought to be essential. We conclude that bromobenzene is a novel inducer of heme oxygenase activity in the liver, differing from other nonheme substances in potency and specificity for the liver, and in utilizing mechanism(s) which require neither production of hepatotoxicity, depletion of hepatic glutathione, nor sensitivity to actinomycin D.     

Effect of cobalt chloride on the activity of key enzymes of heme metabolism in the rat liver

The effects of actinomycin D and cycloheximide on the original and CoCl2-induced delta-aminolevulinate-synthase and heme-oxygenase activities in rat liver were investigated. It was shown that 1.5 hours after CoCl2 administration the delta-aminolevulinate-synthase activity diminishes, then increases up to the original level within the subsequent 4.5 hours, showing a further increase thereafter. The heme-oxygenase activity does not change within 1,5 hours and is then increased. Actinomycin D and cycloheximide suppress the increase in the CoCl2-induced heme-oxygenase activity, whereas that of the delta-aminolevulinate-synthase activity is blocked only by cycloheximide. Hence, the increase in the CoCl2-induced delta-aminolevulinate-synthase activity in the liver is a result of activation of translation. The degree of tryptophan pyrrolase saturation with heme decreases already by the 6th hour, whereas the level of heme in liver mitochondria and microsomes decreases only by the 15th hour following CoCl2 injection. The heme content in the liver shows a further decrease irrespective of the increase in the delta-aminolevulinate synthase activity induced by CoCl2. It may be concluded that under the given experimental conditions this enzyme is not a rate-limiting step in the terminal reaction of heme biosynthesis.     

Heme Inhibition of [delta]-Aminolevulinic Acid Synthesis Is Enhanced by Glutathione in Cell-Free Extracts of Chlorella

In plants, algae, and many bacteria, the heme and chlorophyll precursor, [delta]-aminolevulinic acid (ALA), is synthesized from glutamate in a reaction involving a glutamyl-tRNA intermediate and requiring ATP and NADPH as cofactors. In particulate-free extracts of algae and chloroplasts, ALA synthesis is inhibited by heme. Inclusion of 1.0 mM glutathione (GSH) in an enzyme and tRNA extract, derived from the green alga Chlorella vulgaris, lowered the concentration of heme required for 50% inhibition approximately 10-fold. The effect of GSH could not be duplicated with other reduced sulfhydryl compounds, including mercaptoethanol, dithiothreitol, and cysteine, or with imidazole or bovine serum albumin, which bind to heme and dissociate heme dimers. Absorption spectroscopy indicated that heme was fully reduced in incubation medium containing dithiothreitol, and addition of GSH did not alter the heme reduction state. Oxidized GSH was as effective in enhancing heme inhibition as the reduced form. Co-protoporphyrin IX inhibited ALA synthesis nearly as effectively as heme, and 1.0 mM GSH lowered the concentration required for 50% inhibition approximately 10-fold. Because GSH did not influence the reduction state of heme in the incubation medium, and because GSH could not be replaced by other reduced sulfhydryl compounds or ascorbate, the effect of GSH cannot be explained by action as a sulfhydryl protectant or heme reductant. Preincubation of enzyme extract with GSH, followed by rapid gel filtration, could not substitute for inclusion of GSH with heme during the reaction. The results suggest that GSH must specifically interact with the enzyme extract in the presence of the inhibitor to enhance the inhibition.