Validation of Putative Apicoplast-Targeting Drugs Using a Chemical Supplementation Assay in Cultured Human Malaria Parasites.

Malaria parasites contain a relict plastid, the apicoplast, which is considered an excellent drug target due to its bacterial-like ancestry. Numerous parasiticidals have been proposed to target the apicoplast, but few have had their actual targets substantiated. Isopentenyl pyrophosphate (IPP) production is the sole required function of the apicoplast in the blood stage of the parasite life cycle, and IPP supplementation rescues parasites from apicoplast-perturbing drugs. Hence, any drug that kills parasites when IPP is supplied in culture must have a nonapicoplast target. Here, we use IPP supplementation to discriminate whether 23 purported apicoplast-targeting drugs are on- or off-target. We demonstrate that a prokaryotic DNA replication inhibitor (ciprofloxacin), several prokaryotic translation inhibitors (chloramphenicol, doxycycline, tetracycline, clindamycin, azithromycin, erythromycin, and clarithromycin), a tRNA synthase inhibitor (mupirocin), and two IPP synthesis pathway inhibitors (fosmidomycin and FR900098) have apicoplast targets. Intriguingly, fosmidomycin and FR900098 leave the apicoplast intact, whereas the others eventually result in apicoplast loss. Actinonin, an inhibitor of bacterial posttranslational modification, does not produce a typical delayed-death response but is rescued with IPP, thereby confirming its apicoplast target. Parasites treated with putative apicoplast fatty acid pathway inhibitors could not be rescued, demonstrating that these drugs have their primary targets outside the apicoplast, which agrees with the dispensability of the apicoplast fatty acid synthesis pathways in the blood stage of malaria parasites. IPP supplementation provides a simple test of whether a compound has a target in the apicoplast and can be used to screen novel compounds for mode of action.

Influence of culture conditions on porphyrin production in Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis.

BACKGROUND: Increasing antibiotic resistance among pathogens has raised the demands for new treatment methods such as antimicrobial photodynamic therapy (aPDT) and phototherapy (PT). Experiments for investigating the effects of these methods are often performed in vitro, but the procedures for cultivation of microbes vary between different studies. The aim of this study has been to elucidate how the profile of endogenously produced porphyrins differs by changing the variables of bacteria culturing conditions. METHODS: Two oral pathogens, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, were selected as model organisms. The contents of porphyrins and heme in the bacteria were analysed with liquid chromatography-tandem mass spectrometry when bacteria was cultivated for different lengths of time (3-9 days), upon passaging as well as when growth medium were supplemented with or without horse blood. RESULTS: Both porphyrin and heme content in A. actinomycetemcomitans are highly affected by the age of the culture, and that the porphyrin profiles changes during cultivation. When cultivated colonies of A. actinomycetemcomitans were passaged onto a new, fresh growth medium a large change in porphyrin content occurred. Additional porphyrins were detected; uroporphyrin and 7-carboxylporphyrin, and the total porphyrin content increased up to 28 times. When P. gingivalis was grown on blood containing medium higher concentrations of protoporphyrin IX (2.5 times) and heme (5.4 times) were quantified compared to bacteria grown without blood. CONCLUSIONS: This study demonstrate that there is a need for more standardized culturing protocols when performing aPDT and PT experiments in vitro to avoid large variations in porphyrin profiles and concentrations, the aPDT/PT target compounds, depending on the culturing conditions.

Experiences and concerns of patients with recurrent attacks of acute hepatic porphyria: A qualitative study.

BACKGROUND: The acute hepatic porphyrias (AHPs) are rare inborn errors of heme biosynthesis, characterized clinically by life-threatening acute neurovisceral attacks. Patients with recurrent attacks have a decreased quality of life (QoL); however, no interactive assessment of these patients' views has been reported. We conducted guided discussions regarding specific topics, to explore patients' disease experience and its impact on their lives. METHODS: Sixteen AHP patients experiencing acute attacks were recruited to moderator-led online focus groups. Five groups (3-4 patients each) were conducted and thematic analyses to identify, examine, and categorize patterns in the data was performed. RESULTS: All patients identified prodromal symptoms that began days prior to acute severe pain; the most common included confusion ("brain fog"), irritability, and fatigue. Patients avoided hospitalization due to prior poor experiences with physician knowledge of AHPs or their treatment. All patients used complementary and alternative medicine treatments to avoid hospitalization or manage chronic pain and 81% reported varying degrees of effectiveness. All patients indicated their disease impacted personal relationships due to feelings of isolation and difficulty adjusting to the disease's limitations. CONCLUSION: Patients with recurrent attacks recognize prodromal warning symptoms, attempt to avoid hospitalization, turn to alternative treatments, and have markedly impaired QoL. Counseling and individualized support is crucial for AHP patients with recurrent attacks.

Carbon Fixation Driven by Molecular Hydrogen Results in Chemolithoautotrophically Enhanced Growth of Helicobacter pylori.

UNLABELLED: A molecular hydrogen (H2)-stimulated, chemolithoautotrophic growth mode for the gastric pathogen Helicobacter pylori is reported. In a culture medium containing peptides and amino acids, H2-supplied cells consistently achieved 40 to 60% greater growth yield in 16 h and accumulated 3-fold more carbon from [(14)C]bicarbonate (on a per cell basis) in a 10-h period than cells without H2 Global proteomic comparisons of cells supplied with different atmospheric conditions revealed that addition of H2 led to increased amounts of hydrogenase and the biotin carboxylase subunit of acetyl coenzyme A (acetyl-CoA) carboxylase (ACC), as well as other proteins involved in various cellular functions, including amino acid metabolism, heme synthesis, or protein degradation. In agreement with this result, H2-supplied cells contained 3-fold more ACC activity than cells without H2 Other possible carbon dioxide (CO2) fixation enzymes were not up-expressed under the H2-containing atmosphere. As the gastric mucus is limited in carbon and energy sources and the bacterium lacks mucinase, this new growth mode may contribute to the persistence of the pathogen in vivo This is the first time that chemolithoautotrophic growth is described for a pathogen. IMPORTANCE: Many pathogens must survive within host areas that are poorly supplied with carbon and energy sources, and the gastric pathogen Helicobacter pylori resides almost exclusively in the nutritionally stringent mucus barrier of its host. Although this bacterium is already known to be highly adaptable to gastric niches, a new aspect of its metabolic flexibility, whereby molecular hydrogen use (energy) is coupled to carbon dioxide fixation (carbon acquisition) via a described carbon fixation enzyme, is shown here. This growth mode, which supplements heterotrophy, is termed chemolithoautotrophy and has not been previously reported for a pathogen.

Intersections of pathways involving biotin and iron relative to therapeutic mechanisms for progressive multiple sclerosis.

While there are a variety of therapies for relapsing remitting multiple sclerosis (MS), there is a lack of treatments for progressive MS. An early study indicated that high dose biotin therapy has beneficial effects in approximately 12-15% of patients with progressive MS. The mechanisms behind the putative improvements seen with biotin therapy are not well understood, but have been postulated to include: 1) improving mitochondrial function which is impaired in MS, 2) increasing synthesis of lipids and cholesterol to facilitate remyelination, and 3) affecting gene expression. We suggest one reason that a greater percentage of patients with MS didn't respond to biotin therapy is the inaccessibility or lack of other nutrients, such as iron. In addition to biotin, iron (or heme) is necessary for energy production, biosynthesis of cholesterol and lipids, and for some protective mechanisms. Both biotin and iron are required for myelination during development, and by inference, remyelination. However, iron can also play a role in the pathology of MS. Increased deposition of iron can occur in some CNS structures possibly promoting oxidative damage while low iron levels can occur in other areas. Thus, the potential, detrimental effects of iron need to be considered together with the need for iron to support metabolic demands associated with repair and/or protective processes. We propose the optimal utilization of iron may be necessary to maximize the beneficial effects of biotin. This review will examine the interactions between biotin and iron in pathways that may have therapeutic or pathogenic implications for MS.

Molecular insights into frataxin-mediated iron supply for heme biosynthesis in Bacillus subtilis.

Iron is required as an element to sustain life in all eukaryotes and most bacteria. Although several bacterial iron acquisition strategies have been well explored, little is known about the intracellular trafficking pathways of iron and its entry into the systems for co-factor biogenesis. In this study, we investigated the iron-dependent process of heme maturation in Bacillus subtilis and present, for the first time, structural evidence for the physical interaction of a frataxin homologue (Fra), which is suggested to act as a regulatory component as well as an iron chaperone in different cellular pathways, and a ferrochelatase (HemH), which catalyses the final step of heme b biogenesis. Specific interaction between Fra and HemH was observed upon co-purification from crude cell lysates and, further, by using the recombinant proteins for analytical size-exclusion chromatography. Hydrogen-deuterium exchange experiments identified the landscape of the Fra/HemH interaction interface and revealed Fra as a specific ferrous iron donor for the ferrochelatase HemH. The functional utilisation of the in vitro-generated heme b co-factor upon Fra-mediated iron transfer was confirmed by using the B. subtilis nitric oxide synthase bsNos as a metabolic target enzyme. Complementary mutational analyses confirmed that Fra acts as an essential component for maturation and subsequent targeting of the heme b co-factor, hence representing a key player in the iron-dependent physiology of B. subtilis.

Role of delta-aminolevulinic acid in the symptoms of acute porphyria.

BACKGROUND: Attacks of neuropathic pain, usually abdominal, are characteristic of the acute porphyrias and accompanied by overproduction of heme-precursor molecules, specifically delta-aminolevulinic acid and porphobilinogen. The basis for the acute symptoms in these diseases has been speculative. METHODS: We review genetic acute porphyria, hereditary tyrosinemia, and an acquired condition, lead poisoning. All perturb heme synthesis and present with a similar pain syndrome. RESULTS: Although each of these conditions has characteristic urine biochemistry, all exhibit excess delta-aminolevulinic acid. Moreover, in all, treatment with hemin reduces delta-aminolevulinic acid and relieves symptoms. In contrast, use of recombinant porphobilinogen deaminase to knock down porphobilinogen in acute porphyria was ineffective. CONCLUSIONS: There is now convincing evidence that delta-aminolevulinic acid is the cause of pain in the acute porphyrias. The efficacy of hemin infusion is due mainly, if not entirely, to its inhibition of hepatic delta-aminolevulinic acid synthase-1, the enzyme that catalyzes delta-aminolevulinic acid formation. Delta-aminolevulinic acid synthase-1 is a rational target for additional therapies to control symptoms in acute porphyria.

Effect of 5-aminolevulinic acid on erythropoiesis: a preclinical in vitro characterization for the treatment of congenital sideroblastic anemia.

Congenital sideroblastic anemia (CSA) is a hereditary disorder characterized by microcytic anemia and bone marrow sideroblasts. The most common form of CSA is attributed to mutations in the X-linked gene 5-aminolevulinic acid synthase 2 (ALAS2). ALAS2 is a mitochondrial enzyme, which utilizes glycine and succinyl-CoA to form 5-aminolevulinic acid (ALA), a crucial precursor in heme synthesis. Therefore, ALA supplementation could be an effective therapeutic strategy to restore heme synthesis in CSA caused by ALAS2 defects. In a preclinical study, we examined the effects of ALA in human erythroid cells, including K562 cells and human induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells. ALA treatment resulted in significant dose-dependent accumulation of heme in the K562 cell line. Concomitantly, the treatment substantially induced erythroid differentiation as assessed using benzidine staining. Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed significant upregulation of heme-regulated genes, such as the globin genes [hemoglobin alpha (HBA) and hemoglobin gamma (HBG)] and the heme oxygenase 1 (HMOX1) gene, in K562 cells. Next, to investigate the mechanism by which ALA is transported into erythroid cells, quantitative RT-PCR analysis was performed on previously identified ALA transporters, including solute carrier family 15 (oligopeptide transporter), member (SLC15A) 1, SLC15A2, solute carrier family 36 (proton/amino acid symporter), member (SLC36A1), and solute carrier family 6 (neurotransmitter transporter), member 13 (SLC6A13). Our analysis revealed that SLC36A1 was abundantly expressed in erythroid cells. Thus, gamma-aminobutyric acid (GABA) was added to K562 cells to competitively inhibit SLC36A1-mediated transport. GABA treatment significantly impeded the ALA-mediated increase in the number of hemoglobinized cells as well as the induction of HBG, HBA, and HMOX1. Finally, small-interfering RNA-mediated knockdown of ALAS2 in HiDEP cells considerably decreased the expression of HBA, HBG, and HMOX1, and these expression levels were rescued with ALA treatment. In summary, ALA appears to be transported into erythroid cells mainly by SLC36A1 and is utilized to generate heme. ALA may represent a novel therapeutic option for CSA treatment, particularly for cases harboring ALAS2 mutations.

The role of coproporphyrinogen III oxidase and ferrochelatase genes in heme biosynthesis and regulation in Aspergillus niger.

Heme is a suggested limiting factor in peroxidase production by Aspergillus spp., which are well-known suitable hosts for heterologous protein production. In this study, the role of genes coding for coproporphyrinogen III oxidase (hemF) and ferrochelatase (hemH) was analyzed by means of deletion and overexpression to obtain more insight in fungal heme biosynthesis and regulation. These enzymes represent steps in the heme biosynthetic pathway downstream of the siroheme branch and are suggested to play a role in regulation of the pathway. Based on genome mining, both enzymes deviate in cellular localization and protein domain structure from their Saccharomyces cerevisiae counterparts. The lethal phenotype of deletion of hemF or hemH could be remediated by heme supplementation confirming that Aspergillus niger is capable of hemin uptake. Nevertheless, both gene deletion mutants showed an extremely impaired growth even with hemin supplementation which could be slightly improved by media modifications and the use of hemoglobin as heme source. The hyphae of the mutant strains displayed pinkish coloration and red autofluorescence under UV indicative of cellular porphyrin accumulation. HPLC analysis confirmed accumulation of specific porphyrins, thereby confirming the function of the two proteins in heme biosynthesis. Overexpression of hemH, but not hemF or the aminolevulinic acid synthase encoding hemA, modestly increased the cellular heme content, which was apparently insufficient to increase activity of endogenous peroxidase and cytochrome P450 enzyme activities. Overexpression of all three genes increased the cellular accumulation of porphyrin intermediates suggesting regulatory mechanisms operating in the final steps of the fungal heme biosynthesis pathway.

Malaria parasite-synthesized heme is essential in the mosquito and liver stages and complements host heme in the blood stages of infection.

Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme de novo, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, δ-aminolevulinate synthase (ALAS), and the last enzyme, ferrochelatase (FC), in the heme-biosynthetic pathway of Plasmodium berghei (Pb). The wild-type and knockout (KO) parasites had similar intraerythrocytic growth patterns in mice. We carried out in vitro radiolabeling of heme in Pb-infected mouse reticulocytes and Plasmodium falciparum-infected human RBCs using [4-(14)C] aminolevulinic acid (ALA). We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the de novo pathway is absolutely essential for parasite development in the mosquito and liver stages. PbKO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in PbALASKO parasites recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.

ATP-binding cassette B10 regulates early steps of heme synthesis.

RATIONALE: Heme plays a critical role in gas exchange, mitochondrial energy production, and antioxidant defense in cardiovascular system. The mitochondrial transporter ATP-binding cassette (ABC) B10 has been suggested to export heme out of the mitochondria and is required for normal hemoglobinization of erythropoietic cells and protection against ischemia-reperfusion injury in the heart; however, its primary function has not been established. OBJECTIVE: The aim of this study was to identify the function of ABCB10 in heme synthesis in cardiac cells. METHODS AND RESULTS: Knockdown of ABCB10 in cardiac myoblasts significantly reduced heme levels and the activities of heme-containing proteins, whereas supplementation with δ-aminolevulinic acid reversed these defects. Overexpression of mitochondrial δ-aminolevulinic acid synthase 2, the rate-limiting enzyme upstream of δ-aminolevulinic acid export, failed to restore heme levels in cells with ABCB10 downregulation. ABCB10 and heme levels were increased by hypoxia, and reversal of ABCB10 upregulation caused oxidative stress and cell death. Furthermore, ABCB10 knockdown in neonatal rat cardiomyocytes resulted in a significant delay of calcium removal from the cytoplasm, suggesting a relaxation defect. Finally, ABCB10 expression and heme levels were altered in failing human hearts and mice with ischemic cardiomyopathy. CONCLUSIONS: ABCB10 plays a critical role in heme synthesis pathway by facilitating δ-aminolevulinic acid production or export from the mitochondria. In contrast to previous reports, we show that ABCB10 is not a heme exporter and instead is required for the early mitochondrial steps of heme biosynthesis.

Sampangine inhibits heme biosynthesis in both yeast and human.

The azaoxoaporphine alkaloid sampangine exhibits strong antiproliferation activity in various organisms. Previous studies suggested that it somehow affects heme metabolism and stimulates production of reactive oxygen species (ROS). In this study, we show that inhibition of heme biosynthesis is the primary mechanism of action by sampangine and that increases in the levels of reactive oxygen species are secondary to heme deficiency. We directly demonstrate that sampangine inhibits heme synthesis in the yeast Saccharomyces cerevisiae. It also causes accumulation of uroporphyrinogen and its decarboxylated derivatives, intermediate products of the heme biosynthesis pathway. Our results also suggest that sampangine likely works through an unusual mechanism-by hyperactivating uroporhyrinogen III synthase-to inhibit heme biosynthesis. We also show that the inhibitory effect of sampangine on heme synthesis is conserved in human cells. This study also reveals a surprising essential role for the interaction between the mitochondrial ATP synthase and the electron transport chain.

Oral supplementation of gossypin during lead exposure protects alteration in heme synthesis pathway and brain oxidative stress in rats.

OBJECTIVE: The objective was to study the efficacy of oral supplementation of gossypin, a flavonoid, during lead exposure in preventive alterations in the heme synthesis pathway, brain oxidation, and tissue lead uptake in rats. METHODS: Male rats were used for the experiment and were exposed to lead (0.5% in drinking water) or lead plus oral supplementation of gossypin (25 or 100mg/kg) for 3 wk to determine the preventive effect of gossypin against lead toxicity. Animals were sacrificed after 3 wk for various biochemical variables suggestive of oxidative stress and heme synthesis pathway in addition to the concentration of lead in the blood and brain. RESULTS: Exposure to lead produced significant inhibition in the activity of blood delta-aminolevulinic acid dehydratase accompanied by an increase in urinary delta-aminolevulinic acid and the levels of reactive oxygen species. There were significant alterations in the levels of glutathione, thiobarbituric acid-reactive substances, reactive oxygen species, and superoxide dismutase activity on lead exposure. Most of these alterations were significantly prevented by oral coadministration of gossypin, particularly at the dose of 100mg/kg. CONCLUSION: The antioxidant and moderate chelating properties of oral gossypin suggest a promising role in use either as a nutritional supplement during lead exposure or as a complementary chelating agent during chelation therapy.

Effects of an onion by-product on bioactivity and safety markers in healthy rats.

Onions are excellent sources of bioactive compounds including fructo-oligosaccharides (FOS) and polyphenols. An onion by-product was characterised in order to be developed as a potentially bioactive food ingredient. Our main aim was to investigate whether the potential health and safety effects of this onion by-product were shared by either of two derived fractions, an extract containing the onion FOS and polyphenols and a residue fraction containing mainly cell wall materials. We report here on the effects of feeding these products on markers of potential toxicity, protective enzymes and gut environment in healthy rats. Rats were fed during 4 weeks with a diet containing the products or a control feed balanced in carbohydrate. The onion by-product and the extract caused anaemia as expected in rodents for Allium products. No other toxicity was observed, including genotoxicity. Glutathione reductase (GR) and glutathione peroxidase (GPx1) activities in erythrocytes increased when rats were fed with the onion extract. Hepatic gene expression of Gr, Gpx1, catalase, 5-aminolevulinate synthase and NAD(P)H:quinone oxidoreductase was not altered in any group of the onion fed rats. By contrast, gamma-glutamate cysteine ligase catalytic subunit gene expression was upregulated but only in rats given the onion residue. The onion by-products as well as the soluble and insoluble fractions had prebiotic effects as evidenced by decreased pH, increased butyrate production and altered gut microbiota enzyme activities. In conclusion, the onion by-products have no in vivo genotoxicity, may support in vivo antioxidative defence and alter the functionality of the rat gut microbiota.

Potential application of the recombinant Escherichia coli-synthesized heme as a bioavailable iron source.

To investigate the potential use of microbial heme as an iron source, recombinant Escherichia coli coexpressing ALA synthase (HemA) as well as the NADP-dependent malic enzyme (MaeB) and dicarboxylic acid transporter (DctA) were cultured. The typical red pigment extracted from the recombinant E. coli after 38 h showed highest absorbance at 407 nm, and the amount of iron in 38.4 mg of microbial heme extract derived from 6-l fermentation broth was 4.1 mg. To determine the commercial potential of the recombinant E.coli-synthesized iron-associated heme as an iron source, mice were fed the iron-free provender with the microbial heme extract. The average body weight reduction of mice fed non-iron provender was 2.3%, whereas no detectable weight loss was evident in mice fed microbial heme addition after 15 days. The heme content of the blood from microbial heme fed mice was 4.2 mg/ml whereas that of controls was 2.4 mg/ml, which implies that the microbial heme could be available for use as an animal iron source.

The heme biosynthetic pathway of the obligate Wolbachia endosymbiont of Brugia malayi as a potential anti-filarial drug target.

BACKGROUND: Filarial parasites (e.g., Brugia malayi, Onchocerca volvulus, and Wuchereria bancrofti) are causative agents of lymphatic filariasis and onchocerciasis, which are among the most disabling of neglected tropical diseases. There is an urgent need to develop macro-filaricidal drugs, as current anti-filarial chemotherapy (e.g., diethylcarbamazine [DEC], ivermectin and albendazole) can interrupt transmission predominantly by killing microfilariae (mf) larvae, but is less effective on adult worms, which can live for decades in the human host. All medically relevant human filarial parasites appear to contain an obligate endosymbiotic bacterium, Wolbachia. This alpha-proteobacterial mutualist has been recognized as a potential target for filarial nematode life cycle intervention, as antibiotic treatments of filarial worms harboring Wolbachia result in the loss of worm fertility and viability upon antibiotic treatments both in vitro and in vivo. Human trials have confirmed this approach, although the length of treatments, high doses required and medical counter-indications for young children and pregnant women warrant the identification of additional anti-Wolbachia drugs. METHODS AND FINDINGS: Genome sequence analysis indicated that enzymes involved in heme biosynthesis might constitute a potential anti-Wolbachia target set. We tested different heme biosynthetic pathway inhibitors in ex vivo B. malayi viability assays and report a specific effect of N-methyl mesoporphyrin (NMMP), which targets ferrochelatase (FC, the last step). Our phylogenetic analysis indicates evolutionarily significant divergence between Wolbachia heme genes and their human homologues. We therefore undertook the cloning, overexpression and analysis of several enzymes of this pathway alongside their human homologues, and prepared proteins for drug targeting. In vitro enzyme assays revealed a approximately 600-fold difference in drug sensitivities to succinyl acetone (SA) between Wolbachia and human 5'-aminolevulinic acid dehydratase (ALAD, the second step). Similarly, Escherichia coli hemH (FC) deficient strains transformed with human and Wolbachia FC homologues showed significantly different sensitivities to NMMP. This approach enables functional complementation in E. coli heme deficient mutants as an alternative E. coli-based method for drug screening. CONCLUSIONS: Our studies indicate that the heme biosynthetic genes in the Wolbachia of B. malayi (wBm) might be essential for the filarial host survival. In addition, the results suggest they are likely candidate drug targets based upon significant differences in phylogenetic distance, biochemical properties and sensitivities to heme biosynthesis inhibitors, as compared to their human homologues.

Serum iron increases with acute induction of hepatic heme oxygenase-1 in mice.

Heme oxygenase (HO)-1 is induced by oxidative stress and protects against oxidant injury. We examined the effect of rapid induction of hepatic HO-1 on serum iron level. Serum iron was approximately doubled within 6 h when HO-1 was induced by phenobarbital treatment of selenium-deficient mice. Blocking heme synthesis with diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) prevented the induction of HO-1 and the rise in serum iron. DDC did not block HO-1 induction by hemin. Inhibition of HO activity by tin protoporphyrin prevented a rise in serum iron that occurred following phorone treatment. These results indicate that heme synthesis or an exogenous source of heme is needed to allow induction of HO-1. Further, they link HO-1 induction with a rise in serum iron, suggesting that the iron resulting from catabolism of heme by HO-1 is released by the liver.

RNA silencing of the mitochondrial ABCB7 transporter in HeLa cells causes an iron-deficient phenotype with mitochondrial iron overload.

X-linked sideroblastic anemia with ataxia (XLSA/A) is caused by defects of the transporter ABCB7 and is characterized by mitochondrial iron deposition and excess of protoporphyrin in erythroid cells. We describe ABCB7 silencing in HeLa cells by performing sequential transfections with siRNAs. The phenotype of the ABCB7-deficient cells was characterized by a strong reduction in proliferation rate that was not rescued by iron supplementation, by evident signs of iron deficiency, and by a large approximately 6-fold increase of iron accumulation in the mitochondria that was poorly available to mitochondrial ferritin. The cells showed an increase of protoporphyrin IX, a higher sensitivity to H(2)O(2) toxicity, and a reduced activity of mitochondrial superoxide dismutase 2 (SOD2), while the activity of mitochondrial enzymes, such as citrate synthase or succinate dehydrogenase, and ATP content were not decreased. In contrast, aconitase activity, particularly that of the cytosolic, IRP1 form, was reduced. The results support the hypothesis that ABCB7 is involved in the transfer of iron from mitochondria to cytosol, and in the maturation of cytosolic Fe/S enzymes. In addition, the results indicate that anemia in XLSA/A is caused by the accumulation of iron in a form that is not readily usable for heme synthesis.