Δ-aminolevulinic Acid Dehydrase Research Articles

Inhibition Effect of Poly(γ-glutamic acid) on Lead-Induced Toxicity in Mice

The objectives of this study were to evaluate the efficiency in treatment of lead-induced intoxication in mice with γ-PGA as chelating agent and compare with the drug (meso-2,3-dimercaptosuccinic acid). The results showed the incorporation of γ-PGA at 200 and 400 mg/kg could reduce the accumulation of lead in the liver, heart, and testis; however, the latter was more effective in decreasing the lead content in the kidney and spleen. Nevertheless, both doses failed to inhibit the lead accumulation in the lung and brain. Additionally, both doses of γ-PGA could reduce TBARs in the kidney and brain, as well as elevate δ-aminolevulinic acid dehydrase (δ-ALAD) activity in blood and decrease glutamic pyruvic transaminase (GPT) and lactic dehydrogenase (LDH) activities in the serum. For hematological parameters, both white blood cells (WBCs) and hematocrite (HCT) were raised by 400 mg/kg of γ-PGA, while for both doses of γ-PGA, a slight decline in hemoglobin (HGB), mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC) was observed, with the red blood cells (RBCs) being unaffected.

Only complete rejoining of DNA strand breaks after UVC allows K562 cell proliferation and DMSO induction of erythropoiesis

DNA strand breaks are early intermediates of the repair of UVC-induced DNA damage, however, since they severely impair cellular activities, their presence should be limited in time. In this study, the effects of incomplete repair of UVC-induced DNA strand breaks are investigated on K562 cell growth and the induction of erythroid differentiation by addition of DMSO to the cell culture medium. The kinetics were followed after UV irradiation by single cell gel electrophoresis, and in total cell population by alkaline or neutral agarose gel electrophoresis. Shortly after exposure, an extensive fragmentation occurred in DNA; DNA double strand breaks were negatively correlated with recovery time for DNA integrity. DNA damage induced by UVC 9 J/m 2 rapidly triggered necrosis in a large fraction of irradiated K562 cells, and only 40% of treated cells resumed growth at a very low rate within 24 h of culture. The addition of DMSO to the culture medium of cells 15 min after UVC, when DNA strand break repair was not yet complete, produced apoptosis in >70% of surviving cells, as determined by TUNEL assay. Conversely, if DMSO was added when the resealing of DNA strand breaks was complete, surviving K562 cells retained full growth capacity, and their progeny underwent erythroid differentiation with normal levels of erythroid proteins, δ-aminolevulinic acid dehydrase and hemoglobin. This study shows that the extent of DNA strand break repair influences cell proliferation and the DMSO induced erythroid program, and the same UVC dose can have opposite effects depending on cellular status.

HEPATIC PORPHYRIAS

The porphyrias are metabolic disorders characterized by abnormal heme biosynthesis with excessive accumulation and excretion of porphyrias or porphyrin precursors. Defects in the enzymes of the heme biosynthetic pathway result in porphyria. Several of the disorders have been classified as hepatic because the major site of the biochemical defect has been localized to the liver. This article describes the enzymes of the heme biosynthetic pathway, the clinical features of the hepatic porphyrias and management of the disorders.

Heme Synthesizing Enzymes ofPlasmodium knowlesi:A Simian Malarial Parasite

Intraerythrocytic stages of cell-freePlasmodium knowlesipossess significant activities of heme biosynthetic enzymes, viz. δ-aminolevulinic acid synthase (δ-ALAS), δ-aminolevulinic acid dehydrase (δ-ALAD), ferrochelatase (FC), and tryptophan pyrrolase (enzyme representing free heme pool). δ-Aminolevulinic acid synthase and FC showed higher activities in schizont than in ring trophozoite stages. Uninfected monkey erythrocytes did not possess the above-mentioned enzyme activities; on the contrary, leucocytes showed detectable enzyme activities. δ-Aminolevulinic acid synthase was not appreciably inhibited by different antimalarials. Succinyl acetone and hemin exhibited a concentration-dependent inhibition of δ-ALAD and δ-ALAS, respectively.

Two periods of sensitivity to mutagens in induced Mel cells with different outcomes

Mouse erythroleukemia (Mel) cells are particularly sensitive to mutagenic agents between 18 and 24 h from the start of induction (Foresti, M.L. Gaudio, G. Geraci and P. Manduca (1986) Inhibition of dimethyl sulfoxide induced erythropoietic differentiation of murine erythroleukemia cells in culture, Cancer Res., 46, 6260–6263). We show here the occurrence of another period of sensitivity during the initial 5 h after the addition of the inducer dimethyl sulfoxide (DMSO) to the culture medium. The sensitivity to the mutagenic action of a sublethal 3-s pulse of UV light (13.5 J/m 2 ) was monitored on the progeny of the irradiated cells at day 5 after the start of induction. The effects were analysed on functions strictly linked to the final expression of the differentiated phenotype: hemoglobin concentration, percent cells producing hemoglobin (%B+), activity of δ-amino levulinic acid dehydrase (ALA-DH) and presence of globins. Each function appeared differently and selectively affected in the progeny of the cells depending on the exact time of irradiation during the period of sensitivity. Specifically, cells irradiated at hour 3 after induction show both hemoglobin concentration and ALA-DH activity values increased by a factor 3 over controls. Cells irradiated at hour 5 show an almost complete halt in cell induction and the other tested functions show minimal values. Cells are nearly insensitive to irradiation at later times, until hour 20, after which a second period of sensitivity with peak value at hour 22 occurs at which time hemoglobin concentration in the progeny of irradiated cells is increased by a factor 3 over controls, ALA-DH activity is increased by a factor 15 while percent B+ value is at its minimum. The differential effects of UV irradiation on Mel cell functions in the first and in the second period of sensitivity to mutagens confirm the hypothesis that the consequences of a mutational event are strictly dependent on the functional state of the cell. The 1–5 h period of sensitivity in which Mel cells fix the effects of the mutagen in their genome corresponds to increased thymidine incorporation not correlated with cell duplication.

Activity of erythrocyte δ-aminolevulinic acid dehydratase in the female cynomolgus monkey ( Macaca fascicularis): kinetic analysis in control and lead-exposed animals

Kinetic analysis of erythrocyte δ-aminolevulinic acid dehydrase (δ-ALAD) from female cynomolgus monkeys revealed differences in pH optimum and Michaelis constants according to their exposure to lead. In vitro incubation of δ-ALAD with 5 mM dithiothreitol (DTT) or 100–200 μM zinc resulted in an enhanced enzyme activity being expressed. These effects were additive. Activation with DTT or zinc resulted in the abolition of pH differences between control and exposed animals and revealed an increased quantity of enzyme in exposed animals. δ-ALAD in control monkeys was observed to be very sensitive to inhibition by lead in vitro with an apparent inhibition constant ( K 1 ) of 0.12 μM. The effect of lead on monkey δ-ALAD enzyme kinetics is similar to that seen with human samples and thus is a useful model for measuring biological response to lead exposure.

Iron Distribution in Belgrade Rat Reticulocytes After Inhibition of Heme Synthesis With Succinylacetone

AbstractWe have used succinylacetone (4,6-dioxoheptanoic acid), a specific inhibitor of δ-aminolevulinic acid dehydrase, to gain insight into the defect in iron metabolism in the Belgrade anemia. The Belgrade rat has an inherited microcytic, hypochromic anemia associated with poor iron uptake into developing erythroid cells. Succinylacetone inhibits heme synthesis, leading to nonheme iron accumulation in mitochondria and cytosol of normal reticulocytes. When succinylacetone is used to inhibit Belgrade heme synthesis, iron from diferric transferrin does not accumulate in the stromal fraction that contains mitochondria, nor does 59Fe accumulate in the nonheme cytosolic fraction. Hence, the defect in the Belgrade rat reticulocyte occurs in the endocytic vesicle or in a step subsequent to iron transit from the vesicle but before the nonheme cytosolic or mitochondrial iron fractions. Therefore, the mutation affects either the release of iron from transferrin or iron transport from the vesicle to the mitochondrion.

Effects of lead on the heme biosynthetic pathway in rat kidney

Exposure of rats to lead in drinking water at concentrations of 500 ppm and 1000 ppm for 3 and 6 months resulted in elevated blood lead levels, formation of kidney intranuclear inclusion bodies, and increased urinary excretion of uroporphyrin and coproporphyrin. The erythrocytic Zn-protoporphyrin was increased in the highest dose group. No significant effects on body weight gain or kidney weight were observed. Renal activity of δ-aminolevulinic acid synthetase (ALAS) was not significantly affected by lead treatment. The renal activity of δ-aminolevulinic acid dehydrase (ALAD) was moderately increased and ferrochelatase activity was significantly decreased. The relatively small effects of chronic lead exposure on renal heme biosynthesis suggests that intracellular complexation of lead with high-affinity renal lead binding proteins (PbBP) and formation of intranuclear inclusions in proximal tubule cells protects this highly sensitive pathway in kidney from lead inhibition in vivo. These data also suggest that the observed increases in urinary porphyrin excretion are primarily due to lead effects on the erythropoietic system.

Effects of succinylacetone on dimethylsulfoxide-mediated induction of heme pathway enzymes in mouse Friend virus-transformed erythroleukemia cells

Heme has been reported to exert a control over its own biosynthesis and to affect the erythroid differentiation process at different sites. In this study, succinylacetone, a powerful inhibitor of δ-aminolevulinic acid dehydrase was used to block heme synthesis and to study the effects of heme depletion on the dimethylsulfoxide (DMSO)-mediated induction of the heme pathway enzymes in Friend virus-transformed erythroleukemia cells. The presence of succinylacetone in the medium during the DMSO treatment (1) potentiates the induction of δ-aminolevulinic acid synthetase (the first enzyme of the pathway) and this effect is reversed by the addition of exogenous hemin; (2) does not affect the induction of δ-aminolevulinic acid dehydrase (the second enzyme); (3) prevents the induction of porphobilinogen deaminase (the third enzyme), since no increase could be detected in either the enzyme activity or the immunoreactive protein and this effect could not be reversed by the addition of exogenous hemin; (4) does not affect the induction of ferrochelatase. The possible role of heme or of intermediate metabolites of the pathway on the induction of these enzymes during the erythroid differentiation process is discussed.

Neurotoxic aspects of porphyrinopathies: Lead and succinylacetone

Neurotoxic effects of heavy metals and polyhalogenated hydrocarbons frequently occur at low levels of exposure, in some cases below those levels where direct toxic actions of these compounds have been demonstrated. Since alterations in heme synthesis are effects common to many of these compounds, the potential role of altered heme synthesis in producing or contributing to their neurotoxicity was studied. Rats with acute and chronic lead exposure were compared to rats whose heme synthesis was inhibited by succinylacetone, as a semichronic model of the hereditary heme synthesis disorder, acute intermittent porphyria. Both treatments produce significant inhibition in activity of the enzyme δ-aminolevulinic acid dehydrase and elevations in the heme precursor δ-aminolevulinic acid (ALA) in tissues and urine. Associated with increased ALA is a significant inhibition of neurotransmission utilizing the amino acid γ-aminobutyric acid (GABA), expressed chemically and behaviorally. The results suggest that in addition to their direct molecular neurotoxicity, porphyrinopathic compounds such as lead may, through altering heme synthesis, adversely affect the brain at low levels of exposure.

δ-Aminolevulinic acid synthesis in a Cyanidium caldarium mutant unable to make chlorophyll a and phycobiliproteins

Wild-type cells of the unicellular rhodophyte, Cyanidium caldarium, synthesize chlorophyll a, phycobiliproteins, and heme from δ-aminolevulinic acid during light-dependent chloroplast development but are unable to make photosynthetic pigments in the dark. C. caldarium, mutant GGB-Y, is an obligate heterotroph which, in the light, produces a chloroplast devoid of photosynthetic pigments. The present investigation has shown that δ-aminolevulinic acid is synthesized in cells of mutant GGB-Y incubated with levulinic acid, a competitive inhibitor of δ-aminolevulinic acid dehydrase (the second enzyme in the porphyrin biosynthetic pathway). In vivo, cells of mutant GGB-Y preferentially incorporated C 1 of glutamate and α-ketoglutarate into the C 5 fragment (formaldehyde) of δ-aminolevulinic acid after alkaline periodate degradation. This suggested that δ-aminolevulinic acid arises directly from the carbon skeleton of glutamate and α-ketoglutaric acid. The pattern of incorporation of C 3, C 4, and C 5 of α-ketoglutarate into the C 1–C 4 (succinic acid) fragment of δ-aminolevulinic acid after alkaline periodate degradation was consistent with the origin of δ-aminolevulinic acid from a five-carbon precursor. C 1 and C 2 of glycine and C 2 and C 3 of succinate were incorporated into both the formaldehyde and succinate fragments of δ-aminolevulinic acid in a manner inconsistent with condensation of glycine and succinyl CoA by δ-aminolevulinic acid synthetase, the rate-limiting enzyme in the porphyrin pathway in animals and bacteria. Extracts of the soluble protein from cells of mutant GGB-Y displayed a Soret band at 410 nm indicating the presence of hemoproteins. This shows that mutant GGB-Y cells synthesize heme. The respiration of radiolabeled glutamate, α-ketoglutarate, and glycine to 14CO 2 is consistent with the existence of mitochondrial cytochromes in cells of mutant GGB-Y and with the ability of the mutant to synthesize δ-aminolevulinic acid. The present results suggest that δ-aminolevulinic acid is synthesized directly from glutamate or α-ketoglutarate and that this is the only process by which the rate-limiting intermediate in the porphyrin pathway is synthesized in C. caldarium. If correct, the rate-limiting, regulative enzyme in the biosynthetic pathway for synthesis of chlorophyll a, bile pigment (phycocyanobilin), and heme must have been completely different in the evolutionary antecedents of modern-day plants and animals.

Inhibition of δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydrase by L-2-amino-4-methoxy- [formula omitted]-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 - 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.

A Study on the δ-Aminolevulinic Acid Dehydrase Activity and Lead in Blood Concentration in Women Workers

A Study on the δ-Aminolevulinic Acid Dehydrase Activity and Lead in Blood Concentration in Women Workers

Selenium and lead: Mutual detoxifying effects

Antagonistic toxic effects of selenium and lead were studied in growing rats. Chronic lead intoxication was produced by cutaneous application of lead naphthenate solution (80–200 mg Pb/kg body weight) for a period of 8 weeks and chronic selenium intoxication was induced by giving 5 ppm, 10 ppm and 15 ppm selenium in drinking water. The growth rate and food consumption of rats receiving selenium in addition to lead approached normal rate while animals treated with only one of them showed hampered growth rate and lower food consumption. The enzymatic activity of δ-aminolevulinic acid dehydrase (ALA-D) in whole blood, liver and kidney and liver P-450 enzyme activity were normal in rats receiving both selenium and lead. The enzymic activities assayed were, however, depressed in the animals receiving either lead or selenium. Assay of lead and selenium in liver, brain, kidney and blood was carried out. Rats receiving both metals and higher concentrations of these metals in the organs studied, as compared to those only receiving one component. The data seem to indicate that the effect of selenium on the toxic effects of lead is similar to its protective role against methylmercury intoxication.

Biosynthesis of δ-aminolevulinic acid in the unicellular rhodophyte, cyanidium caldarium

Levulinic acid, a competitive inhibitor of δ-aminolevulinic acid dehydrase, inhibited chlorophyll-a and phycocyanobilin synthesis by 50% in vivo in the unicellular rhodophyte, Cyanidium caldarium. Inhibition of tetrapyrrole synthesis was accompanied by accumulation of δ-aminolevulinic acid in amounts equivalent to the difference between chlorophyll-a and phycocyanobilin synthesis in control and levulinic aicd treated cells. δ-Aminolevulinic acid produced by levulinic acid treated cells incubated with specifically labeled glycine, α-ketoglutarate and glutamate was degraded in alkaline periodate to the succinic acid (C 1-C 4) and formaldehyde (C 5) derivatives. The distribution of radioactivity in these fragments suggested that the carbon skeleton of δ-aminolevulinic acid is derived directly from α-ketoglutarate and glutamate.

Erythrocyte δ-Aminolevulinic Acid Dehydrase Changes in the Preruminant Calf

Sixteen preruminant, male Holstein calves were tested for erythrocyte δ-aminolevulinic acid dehydrase activity, hemoglobin and erythrocyte count in blood samples taken weekly during the first 13 wk of life. δ-Aminolevulinic acid dehydrase showed a continual increase in activity beginning at wk 3 to a maximal activity at wk 9. Hemoglobin content was decreased by wk 5 and erythrocyte count was reduced by wk 6. δ-Aminolevulinic acid dehydrase enzyme activities increased with age corresponding to changes in hemoglobin and erythrocyte count during early postnatal development of the preruminant calf.

Carbonic anhydrase isozymes, hemoglobin-F and glutathione levels in lead-exposed workers

Red cell carbonic anhydrase B and C isozymes and hemoglobin F were studied by the single radial immunodiffusion technique in lead workers exposed to low concentrations of lead. The lead contents, δ-aminolevulinic acid dehydrase and glutathione levels were simultaneously determined. Carbonic anhydrase B, glutathione and δ-aminolevulinic acid dehydrase activity in lead-exposed workers showed relatively low values compared to those of normal subjects not exposed to occupational lead exposure in the past or present. Subjects with blood lead contents of 30 μg per 100 ml or higher showed a significant increase in hemoglobin-F and a significant decrease in carbonic anhydrase B and glutathione levels. These findings indicate that the syntheses of these red cell components are affected by lead, and hence in addition to δ-aminolevulinic acid dehydrase activity, these components were also found to be of clinical value for the diagnosis of subclinical lead poisoning.

Red blood cell beta-aminolevulinic acid dehydrase activity. An index of body lead burden.

It has been suggested that the measurement of the degree of inhibition of red blood cell (RBC) δ-aminolevulinic acid dehydrase (ALAD) activity is a useful indicator for the detection of children with increased lead absorption. Twenty children had simultaneous measurements of blood lead and RBC-ALAD activity, and then received chelation treatment with edetic acid (EDTA). The inhibition of RBC-ALAD activity was found to be highly correlated with the amount of lead excreted in the urine. In this study, the measurement of RBC-ALAD appeared to be superior to the measurement of blood lead in identifying those children with an increased burden of chelatable lead.

Influence of Lead and other Metals on Fish δ-Aminolevulinate Dehydrase Activity

Exposing mummichog (Fundulus heteroclitus) and winter flounder (Pseudopleuronectes americanus) to sublethal concentrations of lead resulted in reduction of liver and kidney δ-aminolevulinic acid dehydrase activity. The results of this project are expected to afford an index of response to heavy metals in estuarine waters.

HUMAN RESPONSES TO LEAD IN SPECIAL REFERENCES TO PORPHYRIN METABOLISM IN BONE MARROW ERYTHROID CELLS, AND CLINICAL AND LABORATORY STUDY

Enzymatic studies on heme biosynthesis in erythroid cells of bone marrows ob-tained from ten lead workers were carried out after the establishment of methods.A remarkable reduction of the activities of δ-aminolevulinic acid (ALA) dehydrase and heme synthetase, a slight elevation of ALA synthetase activity, and a parallel reduction of 14C-glycine incorporation into heme and globin were observed. Abnor-malities found in hematological examinations and in urinary or erthrocyte porphyrin metabolism were discussed in connection with both the above mentioned results and the human response to lead.Clinical and laboratory examinations on these workers revealed a commonly exist-ing hypertension, ECG abnormalities, reduced renal functions, especially those of renal blood circulation and of renal tubules, EMG abnormalities, a reduced conduction velo-city of long nerves, and elevated concentrations of urea-N and uric acid in serum. The pathogenesis of these findings were also discussed.