Effects of colloidal iron overload on renal and hepatic siderosis and the femur in male rats.

Genetic hemochromatosis is an iron overload disorder, and osteopenic and osteoporotic. Femoral neck bone mineral density (BMD) appears to fall with rising hepatic iron concentrations. A critical role for iron in mediating tissue injury is played via hydroxyl radical formation in nephrotoxicity. We investigated the effects of a colloidal iron overload on renal function, organ siderosis, and femoral bone in male rats. Iron overload reduced body growth, and increased the weights of the liver and spleen. Marked deposition of iron was noted in liver and kidney. Activities of lactate dehydrogenase and alkaline phosphatase were decreased, and the concentrations of blood urea nitrogen and creatinine were increased with the reduction in plasma calcium and inorganic phosphorus levels, i.e. functions of the liver and kidney might be affected by reactive oxygen species such as the superoxide radical, H2O2, and the hydroxyl radical produced by overloaded iron. Damage to the proximal tubular epithelial cells of the kidney and a loss of connectivity of cancellous bone in the epiphysis and of trabecular bone in the metaphysis of the distal femur were observed in iron-overloaded rats with a reduction of femoral bone mineral density, i.e. reabsorption of calcium from the proximal tubular epithelial cells of the kidney might be affected and urinary discharge of calcium might be elevated. It was suggested that iron overload gave rise to osteoporosis combined with renal dysfunction and liver iron overload syndrome.

Heme oxygenase-1: a fundamental guardian against oxidative tissue injuries in acute inflammation.

Free heme contributes as a major threat to the oxidative tissue injuries because it catalyzes the formation of reactive oxygen species. When free heme concentration is increased, it results in the induction of heme oxygenase-1 (HO-1), which then breaks free heme down. As such, HO-1 plays a pivotal role in the protection of tissues from oxidative injuries.

The third case of Doss porphyria (delta-amino-levulinic acid dehydratase deficiency) in Germany.

Delta-aminolevulinic acid dehydratase (ALAD) deficiency porphyria, or Doss porphyria, was first reported in Germany in 1979. Only four bona fide cases of Doss porphyria have been reported to date that were confirmed by immunological and molecular analyses of their ALAD mutations. Here we describe the fifth case of Doss porphyria. A 17-year-old German male suffered from colicky abdominal pain and severe polyneuropathy for 2 years. Urinary delta-aminolevulinic acid (ALA) was increased 32-fold, and coproporphyrin 76-fold compared with the upper limit of their respective normal ranges. Urinary excretion of porphobilinogen (PBG) and uroporphyrin was only slightly increased. Faecal porphyrins were within the normal range. Erythrocyte zinc protoporphyrin concentrations were elevated 5.4-fold. ALAD activity in erythrocytes was decreased to 10% of the normal value, and was not activated by zinc and by dithiothreitol. Blood lead levels were within the normal range, excluding lead poisoning in the proband. Erythrocyte ALAD activity was about one-half of the normal value in both parents, whereas it was normal in the proband's brother. Urinary excretion of ALA, PBG and total porphyrins was within the normal range in both parents and the brother. Molecular genetic studies of the ALAD gene in the proband revealed two base changes, C to A and C to T, both in intron 3 at -11 bp upstream of the exon 3 start site. In addition to the proband, the father carried the (-11)C-to-T, while the mother carried the ALAD gene in the proband's brother. These findings suggest that the observed compound heterozygosity of the ALAD gene may be responsible for Doss porphyria in the proband. The proband was successfully treated with haem arginate infusion. The clinical condition improved, and urinary excretion of ALA and coproporphyrin fell to levels of approximately 50% compared with their pretreatment levels during acute relapses. The haem therapy was continued once weekly for 1 year. At the end of 1 year, urinary ALA and porphyrin levels were significantly lowered, and the proband is now almost free of clinical symptoms.

Heme oxygenase-1: a novel therapeutic target in oxidative tissue injuries.

Oxidative stresses such as oxidant stimuli, inflammation, exposure to xenobiotics, or ionizing irradiation provoke cellular protective responses, principally involving transcriptional activation of genes encoding proteins which participate in the defense against oxidative tissue injuries. Excess of free heme, which is released from hemeproteins under such conditions, may constitute a major threat because it can catalyze the formation of reactive oxygen species (ROS). Exposure of mammalian cells to oxidative stimuli induces heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, as well as a 33-kDa heat shock protein. In various model systems, HO-1 induction confers protection on tissues from further injuries, while the abrogation of its induction accelerates cellular injuries. In this article, we review recent advances in the regulatory mechanism of ho-1 gene expression and the role of HO-1 in various models of experimental oxidative tissue injuries, and its potential therapeutic implications.

Marked developmental changes in heme oxygenase-1 (HO-1) expression in the mouse placenta: correlation between HO-1 expression and placental development.

Heme catabolism during embryonic period is not well understood. It has been suggested that placental heme oxygenase (HO)-1, which is an inducible isoform of the rate-limiting enzyme in the heme degradation pathway, may be involved in supporting normal fetal development. In this study, we examined the distribution of HO-1 protein in the developing mouse embryo, as well as developmental changes of ho-1 gene expression, and the enzyme activity of HO and biliverdin IXalpha reductase (BVR-A) in placenta. Ectoplacental cone in embryonic day (E) 6.5 embryo already showed HO-1 protein expression, which became restricted only to trophoblastic cells after placenta formation was completed on day E14.5. The placenta of E13.5-E14.5 embryos expressed high levels of HO-1 mRNA, which was decreased significantly towards the end of pregnancy. However, HO-1 expression in placenta was significantly higher than uterus throughout the gestational period. In contrast to HO-1, the placental level of BVR-A activity remained low and did not show changes throughout the gestational period. The correlation between HO-1 expression and placental development suggests that HO-1 might be essential for normal embryonic development.

A novel MPL point mutation resulting in thrombopoietin-independent activation.

Thrombopoietin (TPO) and its receptor (MPL) are important regulators of megakaryopoiesis. MPL belongs to a cytokine receptor superfamily. To date, all constitutively active MPL mutants have been artificially constructed with amino acid substitutions in the transmembrane domain or extracellular domain of the protein, and they activate signal transduction pathways in Ba/F3 cells that can also be activated by the normal MPL. In this paper, we report a novel spontaneously occurring mutation of MPL, with an amino acid substitution of Trp(508) to Ser(508) in the intracellular domain of MPL, that induces the factor-independent growth of Ba/F3 cells. Examination of intracellular signaling pathways demonstrated that the mutant MPL protein constitutively activates three distinct signaling pathways, SHC-Ras-Raf-MAPK/JNK, JAK-STAT, and PI3K-Akt-Bad.

Splenomegaly induced by recombinant human granulocyte-colony stimulating factor in rats.

Spontaneous ruptures of the spleen have been observed in donors and patients with malignancy during mobilization of peripheral blood stem cells. Thus, we investigated the morphological and histological alteration of the spleen, and mRNA expression levels and activities of the DNA-synthesizing enzymes thymidylate synthase (TS) and thymidine kinase (TK) in the splenic cells, of rats treated with recombinant human granulocyte-colony stimulating factor (rhG-CSF). Daily injections of rhG-CSF for 5 or 7 days slightly enhanced the splenic weight. Single or 3-day treatment with rhG-CSF markedly enhanced the number of granulocytes in peripheral blood. Expression levels of TS and TK mRNA in the splenic cells were significantly increased 6 hours after rhG-CSF treatment. Early expression of TS and TK mRNA in the splenic cells may indicate a reseeding of hematopoietic cells from the bone marrow. Daily injections with rhG-CSF enhanced the TS and TK activities in the splenic cells. As continuous elevations of DNA-synthesizing enzyme activity and spleno-megaly are suggestive of a possible splenic rupture, the monitoring of peripheral granulocytes and splenic size is necessary during the rhG-CSF treatment.

Heme mediates derepression of Maf recognition element through direct binding to transcription repressor Bach1.

Heme controls expression of genes involved in the synthesis of globins and heme. The mammalian transcription factor Bach1 functions as a repressor of the Maf recognition element (MARE) by forming antagonizing hetero-oligomers with the small Maf family proteins. We show here that heme binds specifically to Bach1 and regulates its DNA-binding activity. Deletion studies demonstrated that a heme-binding region of Bach1 is confined within its C-terminal region that possesses four dipeptide cysteine-proline (CP) motifs. Mutations in all of the CP motifs of Bach1 abolished its interaction with heme. The DNA-binding activity of Bach1 as a MafK hetero-oligomer was markedly inhibited by heme in gel mobility shift assays. The repressor activity of Bach1 was lost upon addition of hemin in transfected cells. These results suggest that increased levels of heme inactivate the repressor Bach1, resulting in induction of a host of genes with MARES:

Decrease in the number of sperm associated with decreased blood testosterone levels in male rats treated with extracts from seven plants consumed by natives of northern Thailand.

Natives of northern Thailand believe that certain plants preserved in alcohol provide sexual elevation although there is no scientific proof. To ascertain whether those plants have any biological effects on reproductive activity, we administered the extracts from seven kinds of plants to male rats. Treatments with these extracts resulted in a decrease in the number of sperm associated with decreased serum testosterone levels and an elevation of serum prolactin levels. The circulating levels of follicle stimulating hormone were only slightly affected by all extracts, although some extracts significantly decreased the luteinizing hormone levels. Thus, the extracts may contain a biologically active substance such as phytoestrogen and lead to a hormonal imbalance.

Highly heterogeneous nature of delta-aminolevulinate dehydratase (ALAD) deficiencies in ALAD porphyria.

The properties of 9 delta-aminolevulinate dehydratase (ALAD) mutants from patients with ALAD porphyria (ADP) were examined by bacterial expression of their complementary DNAs and by enzymologic and immunologic assays. ALADs were expressed as glutathione-S-transferase (GST) fusion proteins in Escherichia coli and purified by glutathione-affinity column chromatography. The GST-ALAD fusion proteins were recognized by anti-ALAD antibodies and were enzymatically active as ALAD. The enzymatic activities of 3 ALAD mutants, K59N, A274T, and V153M, were 69.9%, 19.3%, and 41.0% of that of the wild-type ALAD, respectively, whereas 6 mutants, G133R, K59N/G133R, F12L, R240W, V275M, and delTC, showed little activity (< 8%). These variations generally reflect the phenotype of ALAD in vivo in patients with ADP and indicate that GST-ALAD fusion protein is indeed useful for predicting of the phenotype of ALAD mutants. The location of F12L mutation in the enzyme's molecular structure indicates that its disturbance of the quaternary contact of the ALAD dimer appears to have a significant influence on the enzymatic activity. Mouse monoclonal antibodies to human ALAD were developed that specifically recognized a carboxy terminal portion of ALAD, or other regions in the enzyme. This study represents the first complete analysis of 9 mutants of ALAD identified in ADP and indicates the highly heterogeneous nature of mutations in this disorder.

Preventive effects of a Chinese herbal medicine, hochu-ekki-to, on bone loss in ovariectomized rats.

To evaluate the effects of a traditional Chinese herbal medicine, Hochu-ekki-to [Bu-zong-yi-qi-tang], on the bone loss induced by ovariectomy in rats, ovariectomized female rats of the Sprague-Dawley strain at age 35 weeks were daily given Hochu-ekki-to and/or 17 alpha-ethynylestradiol for 8 weeks by gastric tube and, subsequently, the serum hormone levels and the tibial bone mineral density were measured. Hochu-ekki-to treatment suppressed the ovariectomy-induced reduction of the bone mineral density in the whole and metaphysis of tibia with a slight increase of serum levels of estradiol and progesterone, maintaining bone mineral density values similar to that in the estradiol treated ovariectomized rats, as well as the intact control rats. Hochu-ekki-to is suggested to elevate the serum levels of ovarian hormones slightly and prevent bone loss in ovariectomized rats.

Survival of two patients with severe delta-aminolaevulinic acid dehydratase deficiency porphyria.

The course of delta-aminolaevulinic acid dehydratase activity was studied over the 23 years in erythrocytes of two male patients. The enzyme activity was originally 1-2%, which then increased to approximately 8%, of normal levels several years after clinical manifestation of the acute hepatic porphyria syndrome. Urinary excretions of delta-aminolaevulinic acid and coproporphyrin III were excessively increased in the two patients with compound-heterozygous delta-aminolaevulinic acid dehydratase deficiency porphyria.

Molecular analysis of delta-aminolevulinate dehydratase deficiency in a patient with an unusual late-onset porphyria.

Cloning, expression, and genotype studies of the defective gene for delta-aminolevulinate dehydratase (ALAD) in a patient with an unusual late onset of ALAD deficiency porphyria (ADP) were carried out. This patient was unique in that he developed the inherited disease, together with polycythemia, at the age of 63. ALAD activity in erythrocytes of the patient was less than 1% of the normal control level. ALAD complementary DNA (cDNA) isolated from the patient's Epstein-Barr virus (EBV)-transformed lymphoblastoid cells had 2 base transitions in the same allele, G(177) to C and G(397) to A, resulting in amino acid substitutions K59N and G133R, respectively. It has been verified that the patient had no other ALAD mutations in this and in the other allele. By restriction fragment length polymorphism (RFLP) analysis, all family members of the proband who had one-half ALAD activity compared with the ALAD activity of the healthy control were shown to have the same set of base transitions. Expression of ALAD cDNA in CHO cells revealed that K59N cDNA produced a protein with normal ALAD activity, while G133R and K59N/G133R cDNA produced proteins with 8% and 16% ALAD activity, respectively, compared with that expressed by the wild type cDNA. These findings indicate that while the proband was heterozygous for ALAD deficiency, the G(397) to A transition resulting in the G133R substitution is responsible for ADP, and the clinical porphyria developed presumably due to an expansion of the polycythemic clone in erythrocytes that carried the mutant alad allele.

Touch-free in situ investigation of ancient Egyptian pigments.

Some of the pigments painted on the Funerary Stele of Amenemhat (ca. 2000 B.C.) exhibited at the Egyptian Museum, Cairo and on the walls of the Tomb of Userhat (ca. 1420 B.C.), a rock-cut tomb in Thebes, Egypt, were investigated in situ using both a convenient home-made hand-held type of X-ray diffractometer and a commercial X-ray fluorescence spectrometer in a complementary way under touch-free conditions. CaCO3.3MgCO3 (huntite) was found in the white-painted parts of these two ancient monuments. An arsenic (As)-bearing phase was detected in the yellow-painted parts of the latter monument. The occurrence of huntite in Egypt has not been reported previously.

Preventive effects of sulphasalazine on colorectal carcinogenesis in mice with ulcerative colitis.

Sulphasalazine has been used in the treatment of ulcerative colitis and is known to be a prodrug and split into sulphapyridine and 5-aminosalicylic acid by bacteria in the colon. An increased incidence of colorectal carcinoma is known to occur in patients with ulcerative colitis, which displays a recurrence-remission cycle on colorectal mucosa, i.e., the ulceration and regeneration periods of the colorectal mucosa. Repeated mucosal necrosis-regeneration sequence in chronic ulcerative colitis induced with 3% dextran sulfate sodium led to colorectal carcinogenesis in azoxymethane-pretreated mice. Additive treatment with sulphasalazine normalized the enlarged organs, i.e. liver, spleen and kidney and anemia and leucocytosis induced with 3% dextran sulfate sodium resulted in the reduction of tumorous regions with high-grade dysplasia.

Preventive effects of a herbal medicine on bone loss in rats treated with a GnRH agonist.

The study was designed to evaluate the effects of a traditional Chinese herbal medicine Hochu-ekki-to (Bu-zong-yi-qi-tang), which was composed of 10 herbal medicines and had been used for the treatment of oligospermia and as a postoperative medication in Japan, on bone loss in rats treated with a gonadotropin-releasing hormone (GnRH) agonist. Female rats at 40 weeks of age were divided into 4 groups of 8 rats each. In the three experimental groups, each animal received subcutaneous injections of the long-acting GnRH agonist, buserelin acetate, once every four weeks throughout the experiment. Beginning at 48 weeks of age, the experimental groups were given diets containing conjugated estrogens or Hochu-ekki-to for 8 weeks. The administration of the GnRH agonist reduced the bone mineral density in the whole femur to 91.0% of that in the control group. However, administration of conjugated estrogens and Hochu-ekki-to increased the serum concentrations of estradiol 16.8- and 5.3-fold respectively compared with concentrations in the GnRH agonist-treated group, resulting in the augmentation of the bone mineral density to 110.3% and 106.2% respectively. These findings indicate that Hochu-ekki-to enhances the reduced bone mineral density and causes a slight elevation of the serum estradiol levels in the chemically castrated rats.

Novel molecular defects of the delta-aminolevulinate dehydratase gene in a patient with inherited acute hepatic porphyria.

Cloning and expression of the defective gene for delta-aminolevulinate dehydratase (ALAD) from the second of 2 German patients with ALAD deficiency porphyria (ADP), who had been originally reported by Doss et al. in 1979, were performed. Cloning of cDNAs for the defective ALAD were performed using Epstein-Barr virus (EBV)-transformed lymphoblastoid cells of the proband, and nucleotide sequences of cloned cDNA were determined. Two separate mutations of ALAD cDNA were identified in each ALAD allele. One was G457A, termed "H1," resulting in V153M substitution, while the other was a deletion of 2 sequential bases at T(818) and C(819), termed "H2," resulting in a frame shift with a premature stop codon at the amino acid position of 294. Using allele-specific oligonucleotide hybridization, the mother of the proband was shown to have an H1 defect, while using genomic DNA analysis, the father was shown to have an H2 defect. Expression of H1 cDNA in Chinese hamster ovary cells produced an ALAD protein with only a partial activity (10.65% +/- 1.80% of the normal), while H2 cDNA encoded no significant protein. These data thus demonstrate that the proband was associated with 2 novel molecular defects of the ALAD gene, 1 in each allele, and account for the extremely low ALAD activity in his erythrocytes ( approximately 1% of normal).

Interaction between succinyl CoA synthetase and the heme-biosynthetic enzyme ALAS-E is disrupted in sideroblastic anemia.

The first and the rate-limiting enzyme of heme biosynthesis is delta-aminolevulinate synthase (ALAS), which is localized in mitochondria. There are 2 tissue-specific isoforms of ALAS, erythroid-specific (ALAS-E) and nonspecific ALAS (ALAS-N). To identify possible mitochondrial factors that modulate ALAS-E function, we screened a human bone marrow cDNA library, using the mitochondrial form of human ALAS-E as a bait protein in the yeast 2-hybrid system. Our screening led to the isolation of the beta subunit of human ATP-specific succinyl CoA synthetase (SCS-betaA). Using transient expression and coimmunoprecipitation, we verified that mitochodrially expressed SCS-betaA associates specifically with ALAS-E and not with ALAS-N. Furthermore, the ALAS-E mutants R411C and M426V associated with SCS-betaA, but the D190V mutant did not. Because the D190V mutant was identified in a patient with pyridoxine-refractory X-linked sideroblastic anemia, our findings suggest that appropriate association of SCS-betaA and ALAS-E promotes efficient use of succinyl CoA by ALAS-E or helps translocate ALAS-E into mitochondria.

Hematologic aspects of the porphyrias.

The porphyrias are disorders that can be inherited and acquired, in which the activities of the enzymes of the heme biosynthetic pathway are partially or almost totally deficient. There are 8 enzymes involved in the synthesis of heme, and, with the exception of the first enzyme, an enzymatic defect at every step leads to tissue accumulation and excessive excretion of porphyrins and/or their precursors, such as delta-aminolevulinic acid and porphobilinogen. Whereas heme, the final product of the biosynthetic pathway, is biologically important, porphyrins and their precursors are not only useless but also toxic. Porphyrias can be classified as either photosensitive or neurologic, depending on the type of symptoms, but some porphyrias cause both photosensitive and neurologic symptoms. Alternatively, they can be classified either hepatic or erythropoietic, depending on the principal site of expression of the specific enzymatic defect. The tissue-specific expression of porphyrias is largely due to the tissue-specific control of heme pathway gene expression, particularly at the level of delta-aminolevulinate synthase, the first and the rate-limiting enzyme of heme biosynthesis. In this chapter, hematologic aspects of the erythropoietic porphyrias will be described. The 3 major erythropoietic porphyrias are congenital erythropoietic porphyria (CEP), hepatoerythropoietic porphyria (HEP) and erythropoietic protoporphyria (EPP).