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Heme oxygenase-1, the major inducible isoform of heme oxygenase (HO), can be induced by heme and numerous other physical and chemical factors, many of which cause cellular 'stress'. This has led to the realization that HO-1 is a major highly conserved stress or heat shock protein. Recent work has implicated activation of mitogen-activated protein kinases and other kinases in the mechanism of induction of HO-1, and suggested that signal transduction pathways through tyrosine kinases are involved in induction of HO-1 gene expression by stress inducers. We hypothesized that phenylarsine oxide (PAO), an inhibitor of protein tyrosine phosphatases (PTPs), might up-regulate the HO-1 gene. Here, we show that a remarkably brief (1-15 min) exposure of normal hepatocytes to low concentrations (0.5-3 microM) of PAO produces a marked increase in mRNA and protein of HO-1. This increase is comparable to the level obtained by addition of heme (20 microM), and occurs without producing changes in cellular glutathione levels or stabilization of HO-1 message. Preincubation of cells with inhibitors of protein synthesis decreased the ability of PAO to increase levels of HO-1 mRNA, suggesting that the inductive effect requires de novo protein synthesis. Addition of thiol donors abrogated the PAO-mediated induction of HO-1 in a dose dependent fashion. Addition of genistein, a tyrosine kinase inhibitor, blunted the induction produced by both PAO and heme. After brief incubations with PAO or heme, cell extracts showed comparable increases in levels of protein tyrosine phosphorylation in general, and specifically in ZAP70 kinase. Our results are consistent with the proposition that induction of HO-1 by PAO involves inhibition of specific PTP(s), and that the mechanisms of induction of HO-1 by PAO and by heme may share some common pathways.

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Patients with any of the acute porphyrias may suffer from acute attacks. If these patients are treated with certain drugs, such as barbiturates, the likelihood of developing an attack is increased. Patients treated with antidepressants or benzodiazepine-type anxiolytics also could be placed at increased risk of developing porphyric attacks because little is known about the potential for some of these drugs to induce attacks. Primary cultures of chick embryo liver cells were used to study the effects of selected antidepressants and anxiolytics on porphyrin accumulation. Cells were treated with desferrioxamine (to partially block heme synthesis, simulating conditions encountered in porphyric patients) and increasing concentrations (3.16-1000 microM) of the evaluated drugs. Twenty hours later, porphyrin accumulation was measured. The drugs included four antidepressants and five benzodiazepine-type anxiolytics. The antidepressants bupropion and nefazodone significantly increased porphyrin accumulation when given with desferrioxamine, whereas neither fluoxetine nor paroxetine increased porphyrin accumulation. The benzodiazepine-type anxiolytic agents oxazepam, lorazepam, diazepam, triazolam, and midazolam all significantly increased porphyrin accumulation when given with desferrioxamine. Dose-response studies showed that diazepam, midazolam, and triazolam produced significant increases even at the lowest concentration tested (3.16 microM), whereas lorazepam and oxazepam required higher concentrations (>/=10 microM). These studies suggest that patients with acute porphyrias may be at greater risk for developing porphyric attacks when treated with bupropion or nefazodone compared with fluoxetine or paroxetine, and that the evaluated benzodiazepine derivatives should be administered with caution. Among the latter, low doses of lorazepam and oxazepam may be safer than those of diazepam, midazolam, and triazolam.

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When patients with acute porphyrias are treated with antihypertensives and analgesics, they could be placed at increased risk of developing porphyric attacks, since little is known about the potential for many of these drugs to induce these attacks. We used primary chick embryo liver cells, which maintain intact heme synthesis and regulation, to study the effects of antihypertensives and analgesics on porphyrin accumulation. Cells were treated with desferrioxamine to block heme synthesis partially, simulating conditions encountered in porphyric patients. Typically, cells were treated for 20 hr with the test drugs (3.16 to 1000 microM), along with desferrioxamine. Porphyrins were measured spectrofluorometrically, as uro-, copro,- and protoporphyrin. The evaluated drugs included six antihypertensives (two calcium channel blockers, an angiotensin receptor antagonist, and three inhibitors of angiotensin converting enzyme) and eight analgesics. Of the calcium channel blockers tested, nifedipine greatly increased porphyrin accumulation, whereas diltiazem caused only a slight increase. Losartan (an angiotensin receptor antagonist), captopril, or lisinopril (two angiotensin converting enzyme inhibitors) produced only small increases in porphyrin accumulation. In contrast, enalapril (another angiotensin converting enzyme inhibitor) substantially increased porphyrin accumulation when given in high concentrations. Among the analgesics tested, fentanyl and tramadol produced the highest porphyrin accumulations. Nalbuphine, hydrocodone, oxycodone, and dezocine were moderately or weakly porphyrogenic, whereas buprenorphine and morphine did not increase porphyrin accumulation. These studies suggest that patients with acute porphyrias may be at greater risk for developing porphyric attacks when treated with nifedipine (compared with diltiazem), enalapril (compared with captopril or lisinopril), and tramadol (compared with the other analgesics).

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Some patients with acute hereditary porphyrias have seizures and require anticonvulsant therapy, but many anticonvulsants induce exacerbations of the hepatic porphyrias. Recently, several new anticonvulsants have become available. Among these are gabapentin, vigabatrin, felbamate, lamotrigine, and tiagabine. Little is known about their potential for induction of porphyric attacks. We used a cell culture model of primary chicken embryo liver cells, which maintain intact heme synthesis and regulation, to study the effects of these new anticonvulsants on porphyrin accumulation. Treatment of the cells with deferoxamine (250 microM) led to a partial block in heme synthesis, simulating the conditions encountered in human beings with porphyria. Concomitant exposure of these cells to phenobarbital (2 mM) strongly induced accumulation of porphyrins, serving as a positive control in this model. Cells were treated for 20 hours with increasing doses (3.2 to 1,000 microM) of the newer anticonvulsants, with or without deferoxamine. For most of these anticonvulsants 5 to 100 microM is representative of the concentrations achieved in humans with therapeutic doses. Porphyrins were measured spectrofluorometrically as uro-, copro-, and protoporphyrins. Results were confirmed by high-pressure liquid chromatography. Neither vigabatrin nor gabapentin treatment, with or without deferoxamine, led to any increase in porphyrin accumulation. Similar doses of felbamate (with deferoxamine) led to a marked increase in (mainly proto-) porphyrin levels, qualitatively and quantitatively almost identical to the accumulation produced by phenobarbital. Lamotrigine or tiagabine (with deferoxamine) caused similar porphyrin accumulation. Tiagabine treatment up to 100 microM (with deferoxamine) also resulted in very high levels of predominantly proto-porphyrin. In contrast to the other anticonvulsants tested, tiagabine without deferoxamine led to mild porphyrin accumulation. In the presence of deferoxamine, phenobarbital, felbamate, lamotrigine, or tiagabine, but not gabapentin or vigabatrin, increased levels of the mRNA of ALA synthase, the first and rate-controlling enzyme of porphyrin synthesis. Such enzyme induction is a sine qua non for acute porphyric attacks. We conclude that neither vigabatrin nor gabapentin is porphyrogenic, whereas felbamate, lamotrigine, and, especially, tiagabine lead to much accumulation of porphyrins. The latter three anticonvulsants, therefore, may precipitate or exacerbate acute porphyric attacks in humans. We recommend use of vigabatrin or gabapentin, but not felbamate, lamotrigine, or tiagabine, in patients with acute porphyria and seizures.

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Induction of heme oxygenase (HO) has been proposed as a protective cellular mechanism against oxidative damage. In previous work (Tyrrell et al., Carcinogenesis [1993] 14, 761-765), portions of the 5' promoter region of the human HO-1 gene linked to the reporter gene chloramphenicol acetyl transferase (CAT), had been transiently expressed in HeLa cells. To extend the study of human HO gene expression into primary liver cells, these reporter gene fusion constructs, containing 121 or 1416 base pairs of the untranscribed 5'-upstream sequences of the human HO-1 gene, were used along with pSV beta-Gal plasmid to dually transfect primary cultures of chick embryo liver cells (CELC). The transfected cells were treated with selected metals, heme, phorbol ester, and chemical agents that produce oxidative stress (H2O2 or sodium arsenite). Reporter gene activities were measured 18-20 h later. Our major findings are: (1) these HO-CAT constructs were expressed in CELC; (2) unlike HeLa cells, the expression of CAT was detected in CELC without the need for the SV40 enhancer; (3) sodium arsenite and cobalt chloride induced the expression of the HO-CAT constructs whereas heme had no effect on or decreased CAT expression for all of the transfected constructs; (4) study of endogenous chick HO-1 gene expression in CELC showed that HO-1 responded to sodium arsenite treatment in a dose-dependent fashion, and the response was rapid and transient. We conclude that, in chick liver cell cultures, induction of the HO-1 gene by heme is fundamentally different from that produced by transition metals or sodium arsenite. Furthermore, the results suggest that expression of the HO-1 gene is highly conserved across species.

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Heme oxygenase catalyzes the first and rate-controlling step in heme catabolism. One of the two forms of heme oxygenase (heme oxygenase-1) has been shown to be increased by heme, metals, and in some systems, by certain environmental stresses. However, it remains uncertain whether heme induces hepatic heme oxygenase-1 by a general stress response, or a specific heme-dependent cellular response. The work communicated here explores this issue by examining possible mechanisms whereby heme and other metalloporphyrins induce heme oxygenase-1 in normal liver cells. Primary cultures of chick embryo liver cells were tested for their ability to increase heme oxygenase mRNA after exposure to selected metalloporphyrins (heme, chromium mesoporphyrin, cobalt protoporphyrin and manganese protoporphyrin). The ability of antioxidants to decrease metalloporphyrin-mediated induction of heme oxygenase-1 mRNA was also tested. Our results indicate that: 1) the increase in heme oxygenase-1 mRNA mediated by heme or other metalloporphyrins may involve a short-lived protein(s) since the increase was prevented by several inhibitors of protein synthesis; and 2) in normal liver cells, heme-dependent oxidative stress does not play a key role in the heme-mediated induction of heme oxygenase-1. We conclude that heme and other non-heme metalloporphyrins induce heme oxygenase-1 through a mechanism requiring protein synthesis, not because metalloporphyrins increase cellular oxidative or other stress.

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Hepatic 5-aminolevulinic acid synthase, the first and normally rate-controlling enzyme of heme biosynthesis, is regulated by heme. One of the known mechanisms whereby increased cellular heme regulates 5-aminolevulinic acid synthase is by decreasing the stability of its mRNA. In primary cultures of chick embryo liver cells, we tested whether a decrease in cellular heme might increase 5-aminolevulinic acid synthase mRNA stability and whether heme or other metalloporphyrins could reverse this stabilization. We found that: (a) The stability of 5-aminolevulinic acid synthase mRNA was markedly increased by inhibitors of heme biosynthesis, namely, 4,6-dioxoheptanoic acid or deferoxamine; (b) This increased stability of 5-aminolevulinic acid synthase mRNA was reversed by the addition of heme (10 microM) or by the combination of zinc mesoporphyrin (50 nM), an inhibitor of heme oxygenase, and heme (200 nM); (c) Repression of 5-aminolevulinic acid synthase mRNA levels by zinc mesoporphyrin (10 microM) was due to inhibition of heme oxygenase, rather than a direct, heme-like, effect of zinc mesoporphyrin on 5-aminolevulinic acid synthase mRNA; (d) Among the several non-heme metalloporphyrins tested, only zinc mesoporphyrin and chromium mesoporphyrin significantly decreased 5-aminolevulinic acid synthase mRNA without increasing heme oxygenase mRNA.

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Heme oxygenase catalyzes the degradation of heme into biliverdin, carbon monoxide, and iron. Two forms of this enzyme, heme oxygenase-1 and -2, have been identified; only heme oxygenase-1 is subject to induction by heme, metal ions, and other chemical and physical perturbations (e.g. drugs, oxidants, and heat shock). Primary chick embryo liver cells are widely used for the study of heme metabolism because of their ease of preparation, low cost, and high degree of similarity to human heme metabolism. Nonetheless, this system has some limitations: new cultures must be prepared every week; the resulting cell populations are non-homogeneous; and cells are short-lived, limiting the feasible duration of time course and transfection studies. LMH cells are the first chicken hepatoma cell line to be established. The aim of this study was to characterize the regulation of heme oxygenase-1 in LMH cells, and to compare this regulation to that previously described in primary chick embryo liver cells. The induction of heme oxygenase-1 was assessed by measuring changes in mRNA levels or enzyme activities in response to several treatments, including heme, heavy metals, sodium arsenite, and heat shock, which have been shown to increase the expression of heme oxygenase. Similarities were observed with respect to regulation of heme oxygenase-1 expression in primary hepatocytes and LMH cells. We report the first measurable heat shock response of heme oxygenase-1 in CELC or LMH cells; and show that LMH cells are a useful model for the study of heme oxygenase-1 regulation.

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Metalloporphyrins, including heme and others that inhibit heme oxygenase, are agents with expanding therapeutic potential. Recent results from our laboratory showed that a combination of heme and zinc-mesoporphyrin was remarkably effective in ameliorating biochemical features of acute porphyria. The aim of this study was to assess plasma clearance, tissue distribution and persistence, stability, toxicology and metabolic effects of zinc-mesoporphyrin, after its i.v. administration to rats. After administration of 15 mumol/kg b.wt. of zinc-mesoporphyrin (bound to serum albumin in a 1:1 molar ratio) the metalloporphyrin was rapidly cleared from plasma (half-life 3.6 h) with uptake primarily into liver and spleen, considerably less into the kidney and none detectable into the heart or brain. Hepatic heme oxygenase activity was undetectable for 4 days and less than 50% of control 1 week later. Inhibition of splenic heme oxygenase activity was also substantial but less marked than in the liver. No mortality was observed in any of the treated animals, and there was no detectable effect on gross or microscopic appearance of the liver, spleen, kidneys, heart, lungs or brain. Blood counts and chemistries remained within normal limits. We conclude that single doses of ZnMP-serum albumin are nontoxic, rapidly cleared from the plasma and persist primarily in the liver and spleen where heme oxygenase is inhibited for prolonged periods.

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Mifepristone (RU-486), a potent progesterone receptor antagonist and inducer of cytochromes P-450, is currently in use in Europe, particularly as a post-coital oral contraceptive. Soon it will be available in the United States, as well. Since progesterone has been implicated in the pathogenesis of acute attacks of porphyria, the use of RU-486 or related compounds might be considered in porphyric patients. However, as with other cytochrome P-450 inducers, RU-486 may have the ability to precipitate or exacerbate attacks of acute porphyria. The acute porphyrias in relapse are associated with an increase in activity of delta-aminolevulinic acid synthase, the first and normally rate-controlling enzyme in heme biosynthesis. We have used primary cultures of chick embryo liver cells to test the ability of RU-486 to induce delta-aminolevulinic acid synthase activity and mRNA, cytochromes P-450, porphyrin accumulation, and heme oxygenase. We found that RU-486, at concentrations observed in human plasma after a single oral dose, induced the mRNA and activity of delta-aminolevulinic acid synthase, both by itself and in the presence of deferoxamine, a potent iron chelator that inhibits ferrochelatase. RU-486 and deferoxamine together also produced significant accumulations of protoporphyrin. These results indicate that RU-486 may pose a risk in patients with known acute porphyria and should be used with caution. RU-486 increased the concentration of total cytochrome P-450, and the activity of erythromycin demethylase, an activity specifically catalyzed by cytochrome P-450 3A. Unlike several other porphyrogens (e.g. hydantoins, barbiturates), RU-486 does not produce accumulation of uroporphyrin or induction of heme oxygenase in chick embryo liver cells.

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The acute porphyrias in relapse are commonly treated with intravenous heme infusion to decrease the activity of delta-aminolevulinic acid synthase, normally the rate-controlling enzyme in heme biosynthesis. The biochemical effects of heme treatment are short-lived, probably due in part to heme-mediated induction of heme oxygenase, the rate-controlling enzyme for heme degradation. In this work, selected nonheme metalloporphyrins were screened for their ability to reduce delta-aminolevulinic acid synthase mRNA and induce heme oxygenase mRNA in chick embryo liver cell cultures. Of the metalloporphyrins tested, only zinc-mesoporphyrin reduced delta-aminolevulinic acid synthase mRNA without increasing heme oxygenase mRNA. The combination of zinc-mesoporphyrin and heme, at nanomolar concentrations, decreased delta-aminolevulinic acid synthase mRNA in a dose-dependent manner. The combination of zinc-mesoporphyrin (50 nM) and heme (200 nM) decreased the half-life of the mRNA for delta-aminolevulinic acid synthase from 5.2 to 2.5 h, while a similar decrease was produced by heme (10 microM) alone (2.2 h). The ability of zinc-mesoporphyrin to supplement the reduction of delta-aminolevulinic acid synthase mRNA by heme, in a process similar to that observed with heme alone, provides a rationale for further investigation of this compound for eventual use as a supplement to heme therapy of the acute porphyrias and perhaps other conditions in which heme may be of benefit.

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We characterize a liver cell culture model for acute hepatic porphyrias that recapitulates the biochemical features of the human syndrome. In chick embryo liver cells in primary culture exposed to glutethimide and 4,6-dioxoheptanoic acid, heme alone produced a transient dose-dependent decrease in delta-aminolevulinate synthase and a concomitant increase in heme oxygenase. The addition of low concentrations of zinc-mesoporphyrin (50-200 nM), an inhibitor of heme oxygenase, led to more prolonged decreases in activity of the synthase and to an additive effect with heme. These effects of zinc-mesoporphyrin were associated with prolonged inhibition of heme oxygenase. These results suggest that the treatment of choice of acute porphyric syndromes may be the combination of low doses of heme and zinc-mesoporphyrin or another similarly non-toxic inhibitor of heme oxygenase.

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Recent work showed that the combination of 50 microM glutethimide plus 50 microM ferric nitrilotriacetate (FeNTA) synergistically induces heme oxygenase (HO) activity in cultured chick embryo liver cells (Cable et al., Biochem Biophys Res Commun 168: 176-181, 1990). This synergistic induction is due to increased heme synthesis, which then acts to increase HO gene transcription. The aim of the current studies was to characterize the effects on hepatic heme metabolism of (3,5,5-trimethylhexanoyl)ferrocene (TMH-ferrocene), which causes hepatic iron-loading in rats. Unlike FeNTA, TMH-ferrocene alone maximally induced HO activity at 5-10 microM TMH-ferrocene. At higher concentrations, HO activities declined, as did total cellular protein synthesis. Induction of HO was maximal after a 12-hr exposure to TMH-ferrocene, similar to induction by glutethimide plus FeNTA. The effect of TMH-ferrocene on HO could not be ascribed to greater cellular uptake of iron, since cell-associated iron levels were higher after FeNTA than after TMH-ferrocene treatment. TMH-ferrocene (up to 20 microM) did not induce delta-aminolevulinic acid synthase activity. Uroporphyrin accumulation in cells treated with TMH-ferrocene was minimal, but the combination of TMH-ferrocene and glutethimide caused a synergistic increase in uroporphyrin accumulation, similar to treatment with glutethimide plus FeNTA. 4,6-Dioxoheptanoic acid, an inhibitor of heme synthesis, blocked the induction of HO caused by glutethimide and FeNTA, but did not decrease the induction of HO by TMH-ferrocene. TMH-ferrocene-mediated induction of HO does not appear to be due to lipid peroxidation, since malondialdehyde formation was greater for ferrocene (a structural analog of TMH-ferrocene that does not induce HO) than for TMH-ferrocene. Furthermore, the anti-oxidant, butylated hydroxyanisole, which prevented lipid peroxidation, decreased HO induced by glutethimide plus FeNTA, but butylated hydroxyanisole did not affect HO induced by TMH-ferrocene. We conclude that, unlike the combination of glutethimide plus FeNTA, TMH-ferrocene induces HO activity by a mechanism that is independent of cellular heme synthesis.

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