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.     

Regulation of heme metabolism in rat hepatocytes and hepatocyte cell lines: delta-aminolevulinic acid synthase and heme oxygenase are regulated by different heme-dependent mechanisms

Regulation of delta-aminolevulinic acid (ALA) synthase and heme oxygenase was analyzed in primary rat hepatocytes and in two immortalized cell lines, CWSV16 and CWSV17 cells. ALA synthase was induced by 4,6-dioxohepatnoic acid (4,6-DHA), a specific inhibitor of ALA dehydratase, in all three systems; however, the induction in CWSV17 cells was greater than in either of the other two systems. Therefore, CWSV17 cells were used to explore the regulation of both enzymes by heme and 4,6-DHA. Data obtained from detailed concentration curves demonstrated that 4,6-DHA induced the activity of ALA synthase once ALA dehydratase activity became rate-limiting for heme biosynthesis. Heme induced heme oxygenase activity with increases occurring at concentrations of 10 microM or greater. Heme blocked the 4,6-DHA-dependent induction of ALA synthase with an EC50 of 1.25 microM. Heme-dependent decreases of ALA synthase mRNA levels occurred more quickly and at lower concentrations than heme-dependent increases of heme oxygenase mRNA levels. ALA synthase mRNA remained at reduced levels for extended periods of time, while the increases in heme oxygenase mRNA were much more transient. The drastic differences in concentrations and times at which heme-dependent effects were observed strongly suggest that two-different heme-dependent mechanisms control the ALA synthase and heme oxygenase mRNAs. In CWSV17 cells, heme decreased the stability of ALA synthase mRNA from 2.5 to 1.3 h, while 4,6-DHA increased the stability of the mRNA to 5.2 h. These studies demonstrate that regulation of ALA synthase mRNA levels by heme in a mammalian system is mediated by a change in ALA synthase mRNA stability. The results reported here demonstrate the function of the regulatory heme pool on both ALA synthase and heme oxygenase in a mammalian hepatocyte system.     

Altered regulation of hepatic heme metabolism in cadmium exposed chick embryo

An investigation on the process of heme metabolism with special emphasis on ALA synthetase, heme synthetase and heme oxygenase was studied in cadmium exposed chick embryo to enlighten the mechanism of cadmium embryotoxicity. Cadmium chloride injection (2.5-10 mumole/kg) to chick embryo increases the activity of ALA synthetase by 5-7 folds, however, it inhibits the activity of heme synthetase significantly. The activity of heme oxygenase is further shown to be enhanced by cadmium chloride treatment. These changes are accompanied by a marked reduction in hepatic heme content. The induction of ALA synthetase and heme oxygenase was dependent on the initial concentration of exogenous cadmium. Pretreatment with actinomycin D completely blocks the cadmium mediated induction of both ALA synthetase and heme oxygenase. Time course studies on the stimulation of these two enzymes show that cadmium enhances the activity of heme oxygenase to its maximum level after 24 h. of injection, whereas ALA synthetase activity reaches its highest value only by 48 h. and both the enzymes remain elevated at least upto 96 h. This observation can be correlated with the hepatic heme level at different time intervals after cadmium exposure. These observations suggest the presence of regulatory process for heme metabolism which is susceptible to alteration of 'regulatory heme pool' caused by cadmium.     

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.     

The source of heme for vascular heme oxygenase II: de novo heme biosynthesis in rat aorta

Heme is an essential prosthetic group or substrate for many proteins, including hemoglobin, and hemo enzymes such as nitric oxide synthase, soluble guanylyl cyclase, and heme oxygenase (HO). HO is responsible for the breakdown of heme into equimolar amounts of biliverdin, iron, and carbon monoxide, the latter of which is thought to play a role in the regulation of vascular tone. It is not clear whether the source of heme for cardiovascular functions is derived from uptake from the extracellular milieu or synthesis. In this study, we tested the hypothesis that blood vessels obtain their supply of heme for HO through de novo synthesis. Adult male Sprague-Dawley rat aorta was incubated at 37 degrees C in Krebs' solution with 1 micro M [14C]delta-aminolevulinic acid (ALA). [14C]ALA uptake was linear for about 30 min and reached a plateau at approximately 100 min. The radioactivity was incorporated into porphyrins and heme as determined by esterification of 14C-labelled metabolites and thin-layer chromatography. The first and rate-limiting step of heme biosynthesis is catalyzed by ALA synthase (ALA-S), the activity of which was determined in rat aorta using a radiometric assay, approximately 250 nmol x (g wet mass)(-1) x h(-1). Inducing HO-1 in rat aorta with S-nitroso-N-acetylpenicillamine (500 micro M) did not increase ALA-S activity as compared with basal activity levels of the enzyme. It appears that there is a sufficient amount of heme available under basal ALA-S activity conditions to meet the increased demand for heme resulting from HO-1 induction. These observations indicate that the complete enzymatic pathway for de novo heme biosynthesis resides in rat aorta and furthermore indicate that de novo heme synthesis is capable of supplying a substantial portion of the heme substrate for HO in the aorta.     

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.     

Alterations in hepatic heme metabolism in fish exposed to sublethal cadmium levels

A study on hepatic heme metabolism with special emphasis to ALA synthetase, ALA dehydratase and heme oxygenase was carried out in cadmium exposed freshwater fish Channa punctatus to enlighten the mechanism of cadmium induced toxicity. Cadmium exposure (0.5-5.0 mg/1) for 7 days increased the hepatic level of ALA, along with the depletion in heme content, which are characteristic to chemical porphyria. The resultant enhancement in the activities of ALA synthetase and heme oxygenase were further shown to be dose dependent. ALA dehydratase activity on the other hand was enhanced only at higher exposure. Time course studies on the enzyme activities and heme content showed that ALA synthetase started to increase after 24 hrs., reached maximum at 7 days and came back nearly to normal level after 30 days of exposure. Simultaneously maximum depletion in heme level occurred on 7 days of exposure, tending to return to normal on 30 day. In addition, attempt has been made to correlate alterations in heme metabolism due to cadmium with the histopathological manifestations in liver.     

Interaction of the inhibitor of the 3',5' cyclic AMP phosphodiesterase of Dictyostelium discoideum with the Affi-Gel blue

Disodium ethylenediamine tetraacetic acid and/or allylisopropylacetamide administration to rat pups did not evoke a premature induction of hepatic δ-aminolevulinic acid synthetase. Administration of iron to adult rats did not alter δ-aminolevulinic acid synthetase activity and had little inductive effect on heme oxygenase activity. Both heme and cobalt/dextran rapidly induced microsomal heme oxygenase by 3–8 fold. Induction of heme oxygenase by heme could be totally blocked by concurrent administration of cycloheximide. These results argue against the hypothesis that iron is the physiological mediator of δ-aminolevulinic acid synthetase activity.     

Cobalt regulation of heme synthesis and degradation in avian embryo liver cell culture

Inorganic cobalt was found to induce heme oxygenase activity in primary cultures of embryonic chick liver cells and to inhibit the induction of delta-aminolevulinate synthetase by the porphyrinogenic compounds allylisopropylacetamide, dicarbethoxy-1,4-dihydrocollidine, etiocholanolone, phenobarbital, Aroclor (R)1254, and secobarbital. Much smaller concentrations of Co2+ (5 muM) were required to inhibit delta-aminolevulinate synthetase than to induce heme oxygenase activity (50 muM). These effects of Co2+ on heme synthesis and heme degradation were potentiated by depletion of cellular glutathione content as a result of treatment with diethyl maleate. Cobalt inhibition of the induction of delta-aminolevulinate synthetase was of the same magnitude and probably involved the same mechanism as that produced by cobalt heme dimethyl ester and iron heme. The induction of heme oxygenase by cobalt could be blocked by cycloheximide. Plasma protein synthesis was not inhibited in the presence of concentrations of Co2+ which produced inhibition of delta-aminolevulinate synthetase or induction of heme oxygenase. Other metals such as Cd2+ and Cu2+ also inhibited the induction of delta-aminolevulinate synthetase by allylisopropylacetamide. These findings indicate that Co2+ can regulate heme metabolism directly in liver cells without intermediate actions on extrahepatic tissues. It is suggested that regulation of production of delta-aminolevulinate synthetase and heme oxygenase is mediated through the action of the metal ion rather than the metal in the form of a tetrapyrrole chelate.     

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.     

Cytochrome P-450 heme and the regulation of hepatic heme oxygenase activity

The degradation of cytochrome P-450 heme in the liver has been studied by a new approach. In rats, hepatic heme was labeled by administration of a tracer pulse of [5-¹⁴C]δ-aminolevulinic acid (ALA), and its degradation was analyzed in terms of labeled carbon monoxide (¹⁴CO) excretion, which is a specific degradation product of the labeled heme. Within minutes after administration of [5-¹⁴C]ALA, 14CO was detectable and increased after 2 h to an “early peak,” reflecting the elimination of labeled heme from a rapidly turning over pool in the liver. Beyond the early peak, the rate of ¹⁴CO production decreased in a log-linear manner, consistent with the degradation of heme in stable hepatic hemoproteins. From the rate at which ¹⁴CO production declined during this phase, from the predominant labeling of cytochrome P-450 heme by the administered [5-¹⁴C]ALA and from the known turnover characteristics of this hemoprotein in the liver, it could be inferred that production of ¹⁴CO—between 16 and 30 h after administration of labeled ALA—largely reflected degradation of cytochrome P-450 heme. This approach, which permits serial measurements in a single animal, was used to study the effect on cytochrome P-450 heme of administered heme or endotoxin, both of which are potent stimulators of hepatic heme oxygenase activity. Both of these substances caused marked acceleration of the degradation of cytochrome P-450 heme, the effect occurring over the same dose range as that for stimulation of hepatic heme oxygenase. The findings suggest that stimulation of this enzyme activity in the liver is closely related to the rate of degradation of cytochrome P-450 heme.     

Glutathione depletion and anaesthesia in mice alter heme and drug metabolising enzymes

The effects of enflurane and isoflurane on heme metabolism, its regulation, and on some parameters involved in the hepatic drug metabolising system in animals under GSH depletion were investigated. A single dose of the anaesthethics (1 ml kg(-1), i.p.) was administered to control and GSH depleted mice, animals were sacrificed 20 min after. As a consequence of GSH depletion, a significant inhibition in delta-Aminolevulinic acid synthetase activity, the first enzyme of heme biosynthesis, and a striking induction in Heme oxygenase activity, the main enzyme of heme metabolism, were observed. Cytochrome P-450 levels and the activities of P-4502E1 and glutathione S-transferase were increased. These changes in heme metabolism and drug metabolising enzyme system were not altered further by the administration of enflurane or isoflurane. These findings would indicate that the status of oxidative stress produced by GSH depletion could not be affected by these anaesthetics and/or that disturbances in heme metabolism were already too important to undergo further variations.     

Inhibition of δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydrase by L-2-amino-4-methoxy-trans-3-butenoic acid in the rat

The rate limiting enzyme of heme biosynthesis, δ-aminolevulinic acid synthetase (ALA synthetase), and the second enzyme in the heme biosynthetic pathway, δ-aminolevulinic acid dehydrase (ALA dehydrase), were inhibited by the olefinic amino acid L-2-amino-4-methoxy - $\text{trans}$-3-butenoic acid (AMTB). Administration of AMTB (20 mg/kg; i.p.) to rats inhibited ALA synthetase and ALA dehydrase in control animals and in animals with markedly elevated activity of ALA synthetase which resulted from the administration of 3,5-dicarbethoxy-1,4-dimethyl-collidine (DDC, 200 mg/kg, i.p.) or allylisopropylacetamide (200 mg/kg, s.c.). AMTB also blocked the synthesis of rat hepatic porphyrins and inhibited the increase in the urinary excretion of δ-aminolevulinic acid and porphobilinogen following DDC (150 mg/kg, p.o.) administration. Preincubation of AMTB with liver mitochondria or a soluble fraction of liver decreased the activity of mitochondrial ALA synthetase and soluble ALA dehydrase, respectively.     

Hepatic heme metabolism in selenium-deficient rats: effect of phenobarbital.

Hepatic heme metabolism was examined in selenium (Se)-deficient and Se-adequate (control) rats. Administration of phenobarbital stimulated heme synthesis in the liver in both Se-deficient and Se-adequate rats. In contrast to these results, phenobarbital increased microsomal heme oxygenase (MHO) activity six- to eightfold in Se-deficient but not control rats. These data suggest that the previously reported abnormalities of cytochrome P-450 induction in Se-deficient rats are related to increased degradation of hepatic heme.     

The role of heme metabolism during the induction of hepatic and renal cytochrome P-450 levels and drug-metabolizing enzymes in rats by a Prudhoe Bay crude oil

Administration of Prudhoe Bay crude oil (PBCO) to rats resulted in a dose-related increase in liver weight; rapid and marked increase in the activity of hepatic delta-aminolevulinate synthetase, the initial and rate-limiting enzyme in the heme biosynthetic pathway; rapid decline in the activity of hepatic heme oxygenase, the rate-limiting enzyme of heme catabolism; and more gradual increase in the levels of hepatic cytochrome P-450 and some mixed-function oxidase activities such as benzo[a]pyrene hydroxylase and 7-ethoxyresorufin-O-deethylase. PBCO treatment also increased renal cytochrome P-450 levels and mixed-function oxidase activities; however, delta-aminolevulinate synthetase and heme oxygenase activities were unchanged. This suggests that different regulatory mechanism(s) may be involved in renal heme metabolism and induction of monoxygenase system.     

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.     

Heme degradation by the microsomal heme oxygenase system

Heme degradation in the heme oxygenase reaction proceeds essentially as an autocatalytic oxidation of heme which is bound to heme oxygenase; in this reaction heme acts as both the substrate and the coenzyme which activates molecular oxygen. Synthesis of heme oxygenase can be induced by heme itself, in a substrate-mediated induction.     

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.     

Control of cytochromes P450 expression in Gunn rat liver: implication of the intracellular heme pool

The absence of changes in the overall hepatic cytochrome P450 content after administration of 3-methylcholanthrene (MC) to congenitally jaundiced Gunn rats is believed to be related to a limited heme availability in this strain of rat. The amount of available heme, estimated by tryptophan pyrrolase activity, shows a substantial decrease in control Gunn versus control Wistar rats. This reduction is moderately enhanced by MC treatment in Gunn rats but is abolished after phenobarbital administration. Heme oxygenase activity is diminished in Gunn rats and consequently is not responsible for the decrease in the hepatic heme availability. These data point out that the depletion of the intracellular heme can lead to a limitation in the synthesis of cytochrome P450 isoenzymes in the MC-induced Gunn rat.     

Heme oxygenase and heme degradation

The microsomal heme oxygenase system consists of heme oxygenase (HO) and NADPH-cytochrome P450 reductase, and plays a key role in the physiological catabolism of heme which yields biliverdin, carbon monoxide, and iron as the final products. Heme degradation proceeds essentially as a series of autocatalytic oxidation reactions involving heme bound to HO. Large amounts of HO proteins from human and rat can now be prepared in truncated soluble form, and the crystal structures of some HO proteins have been determined. These advances have greatly facilitated the understanding of the mechanisms of individual steps of the HO reaction. HO can be induced in animals by the administration of heme or several other substances; the induction is shown to involve Bach1, a translational repressor. The induced HO is assumed to have cytoprotective effects. An uninducible HO isozyme, HO-2, has been identified, so the authentic HO is now called HO-1. HOs are also widely distributed in invertebrates, higher plants, algae, and bacteria, and function in various ways according to the needs of individual species.