Inhibition Of Δ-aminolevulinic Acid Dehydratase Research Articles

Low Level of Lead Can Induce Phosphatidylserine Exposure and Erythrophagocytosis: A New Mechanism Underlying Lead-Associated Anemia

Anemia is probably one of the most well-known toxic effects of lead. Previously, lead-induced anemia was considered to be from the inhibition of δ-aminolevulinic acid dehydratase participating in the heme biosynthesis. However, little is known whether lead could affect the destruction of erythrocyte, another important factor for anemia. In the present study, we demonstrated that lead could accelerate the splenic sequestration of erythrocytes through phosphatidylserine (PS) exposure and subsequently increased erythrophagocytosis. In freshly isolated human erythrocytes, Pb(2+)- induced PS exposure at relatively low concentrations (∼0.1 μM) by inhibiting flippase, a key aminophospholipid translocase for the maintenance of PS asymmetry and adenosine triphosphate depletion appeared to underlie this phenomenon. Abnormal shape changes of erythrocytes and microvesicle generation and other triggers for the erythrophagocytosis were also observed in the Pb(2+)-exposed erythrocytes. In vitro data showed that human macrophage indeed recognized and phagocytosis PS-exposed erythrocytes. In good accordance with these in vitro results, the oral administration of Pb(2+) increased PS exposure on erythrocytes in rat in vivo. In addition, reduction of hematocrit and hemoglobin and increased spleen weight were observed along with enhanced splenic sequestration of erythrocytes in the rats exposed to Pb(2+) subchronically for 4 weeks through drinking water. In conclusion, these results suggest that Pb(2+)-induced anemia may be explained at least in part by increased PS exposure on erythrocytes, erythrophagocytosis, and splenic sequestration.

Therapeutic effects of Moringa oleifera on arsenic-induced toxicity in rats

Moringa oleifera Lamarack (English: Horseradish-tree, Drumstick-tree; Hindi: Saijan; Sanskrit: Shigru) belongs to the Moringaceae family, is generally known in the developing world as a vegetable, a medicinal plant and a source of vegetable oil. Besides, the plant is reported to have various biological activities, including hypocholesterolemic agent, regulation of thyroid hormone status, anti-diabetic agent, gastric ulcers, anti-tumor agent and hypotensive agent, used for treating various diseases such as inflammation, cardiovascular and liver diseases. Therapeutic efficacy of oral administration of seed powder of M. oleifera (500 mg/kg, orally, once daily) post arsenic exposure (100 ppm in drinking water for 4 months) was investigated in rats. Animals exposed to arsenic(III) showed a significant inhibition of δ-aminolevulinic acid dehydratase (ALAD) activity, decrease in reduced glutathione (GSH) level and an increase in reactive oxygen species (ROS) in blood. On the other hand, a significant decrease in hepatic ALAD, and an increase in δ-aminolevulinic acid synthetase (ALAS) activity was noted after arsenic exposure. These changes were accompanied by an increase in thiobarbiturc acid reactive substances (TBARS) level in liver and kidney. Activities of liver, kidney and brain superoxide dismutase (SOD) and catalase also showed a decrease on arsenic exposure. Administration of M. oleifera seed powder post arsenic exposure, exhibited significant recovery in blood ALAD activity while, it restored blood GSH and ROS levels. Most of the other blood biochemical variables remained unchanged on M. oleifera supplementation. A significant protection in the altered ALAD and ALAS activities of liver and TBARS level in liver and kidney was however, observed after M. oleifera administration. Interestingly, there was a marginal but significant depletion of arsenic from blood, liver and kidneys. The results, thus lead us to conclude that post arsenic exposure administration with the seed powder of M. oleifera has significant role in protecting animals from arsenic-induced oxidative stress and in the depletion of arsenic concentration. Further studies thus can be recommended for determining the effect of co-administrating seed powder of M. oleifera during chelation therapy with a thiol chelator.

Newborn screening for hepatorenal tyrosinemia by tandem mass spectrometry: analysis of succinylacetone extracted from dried blood spots

Objectives To develop a method for the determination of succinylacetone (SA) in dried blood spots (DBS) using tandem mass spectrometry (MS/MS). Methods SA was extracted from DBS with an acetonitrile and water solution (80:20 by volume) containing formic acid and hydrazine hydrate (both at 0.1% by volume), and analyzed by MS/MS with a total run time per sample under 2 min. The reference range for SA in newborns was determined by analyzing a control group of 3199 DBS. SA was also measured in stored newborn specimens from three patients diagnosed clinically with hepatorenal tyrosinemia (HT). Results The within-run precision was ≤7.4% and total precision was ≤12.2% on blood spots fortified with SA at 2, 10, and 50 μmol/L. The limit of quantitation was 1 μmol/L and the calibration was linear from 1 to 50 μmol/L. A comparison of SA analysis of this MS/MS method with an established enzyme assay indirectly quantitating SA by inhibition of δ-aminolevulinic acid dehydratase demonstrated a strong correlation. The reference range in the control group of unaffected newborn was determined to be <2 μmol/L, while SA in retrieved DBS from HT patients was significantly elevated, measuring 46.7, 36.5, and 23.2 μmol/L. Conclusions We report a simple method that can be used for screening HT in newborns. Based on our limited experience, a 2 μmol/L cutoff could result in up to 100% sensitivity and specificity.

Beneficial effect of combined administration of some naturally occurring antioxidants (vitamins) and thiol chelators in the treatment of chronic lead intoxication

Ameliorative effects of few naturally occurring antioxidants like ascorbic acid (vitamin C), α-tocopherol (vitamin E) either alone or in combination with meso-2,3-dimercaptosuccinic acid (DMSA) or monoisoamyl DMSA (MiADMSA), on parameters indicative of oxidative stress in the liver, kidney, brain and blood of lead-exposed rats were studied. Male Wistar rats were exposed to 0.1% lead acetate in drinking water for 3 months and treated thereafter with DMSA or its analogue MiADMSA (50 mg/kg, intraperitoneally), either individually or in combination with vitamin E (5 mg/kg, intramuscularly) or vitamin C (25 mg/kg, orally) once daily for 5 days. The effects of these treatments in influencing the lead-induced alterations in haem synthesis pathway, hepatic, renal and brain oxidative stress and lead concentration from the soft tissues were investigated. Exposure to lead produced a significant inhibition of δ-aminolevulinic acid dehydratase (ALAD) activity from 8.44±0.26 in control animals to 1.76±0.32 in lead control, reduction in glutathione (GSH) from 3.56±0.14 to 2.57±0.25 and an increase in zinc protoporphyrin level from 62.0±3.9 to 170±10.7 in blood, suggesting altered haem synthesis pathway. Both the thiol chelators and the two vitamins were able to increase blood ALAD activity towards normal, however, GSH level responded favorably only to the two thiol chelators. The most prominent effect on blood ALAD activity was, however, observed when MiADMSA was co-administered with vitamin C (7.51±0.17). Lead exposure produced a significant depletion of hepatic GSH from 4.59±0.78 in control animals to 2.27±0.47 in lead controls and catalase activity from 100±3.4 to 22.1±0.25, while oxidized glutathione (GSSG; 0.34±0.05 to 2.05±0.25), thiobarbituric acid reactive substance (TBARS; 1.70±0.45 to 5.22±0.50) and glutathione peroxidase (GPx) levels (3.41±0.09 to 6.17±0.65) increased significantly, pointing to hepatic oxidative stress. Altered, reduced and oxidized GSH levels showed significant recovery after MiADMSA and DMSA administration while, vitamins E and C were effective in reducing GSSG and TBARS levels and increasing catalase activity. Administration of MiADMSA alone and the combined administration of vitamin C along with DMSA and MiADMSA were most effective in increasing hepatic GSH levels to 4.88±0.14, 4.09±0.12 and 4.30±0.06, respectively. Hepatic catalase also reached near normal level in animals co-administered vitamin C with DMSA or MiADMSA (82.5±4.5 and 84.2±3.5, respectively). Combined treatments with vitamins and the thiol chelators were also able to effectively reduce lead-induced decrease in renal catalase activity and increase in TBARS and GPx level. Combination therapy, however, was unable to provide an effective reversal in the altered parameters indicative of oxidative stress in different brain regions, except in catalase activity. The result also suggests a beneficial role of vitamin E when administered along with the thiol chelators (particularly with MiADMSA) in reducing body lead burden. Blood lead concentration was reduced from 13.3±0.11 in lead control to 0.3±0.01 in MiADMSA plus vitamin E-treated rats. Liver and kidney lead concentration also showed a most prominent decrease in MiADMSA plus vitamin E co-administered rats (5.29±0.16 to 0.63±0.02 and 14.1±0.21 to 1.51±0.13 in liver and kidney, respectively). These results thus suggest that vitamin C administration during chelation with DMSA/MiADMSA was significantly beneficial in reducing oxidative stress however, it had little or no additive effect on the depletion of lead compared with the effect of chelators alone. Thus, the co-administration of vitamin E during chelation treatment with DMSA or MiADMSA could be recommended for achieving optimum effects of chelation therapy.

Lead induced oxidative damage and its response to combined administration of α-lipoic acid and succimers in rats

Alpha-lipoic acid (LA) has been reported to be highly effective in improving the thiol capacity of the cells and in reducing lead induced oxidative stress. These results suggested its possible role as a therapeutic intervention of lead poisoning in combination with a chelator. We investigated the effects of LA, either alone or when administered in combination with succimer (meso 2,3-dimercaptosuccinic acid; DMSA or one of its analogue monoisoamyl DMSA), in influencing the lead induced alterations in haem synthesis pathway, hepatic, renal and brain oxidative stress and lead concentration from blood and soft tissues. The results suggest a significant lead induced inhibition of δ-aminolevulinic acid dehydratase (ALAD), reduction in glutathione (GSH) and an increased zinc protoporphyrin (ZPP) level in blood, indicating altered heme synthesis pathway. Both the thiol chelators were able to increase blood ALAD activity and GSH level towards normal. The most prominent effect on blood ALAD activity was however observed when monoisoamyl DMSA (MiADMSA) was co-administered with LA. Lead exposure produced significant depletion of hepatic GSH, while, oxidized glutahione (GSSG), thiobarbituric acid reactive substances (TBARS) and catalase activity increased significantly, suggesting hepatic oxidative stress. All the treatments were able to increase hepatic GSH and reduce GSSG levels, while, TBARS level reduced significantly in animals administered LA and MiADMSA, individually or in combination. Lead induced increase in renal GSSG, TBARS levels and catalase activity, were effectively reduced by LA, while, the two chelators when administered alone were effective only in reducing GSSG and catalase activity. The most prominent beneficial effects, however, were observed in animals treated concomitantly with LA and one of the chelators (DMSA or MiADMSA). Brain GSH and GSSG levels decreased moderately while superoxide dismutase (SOD) activity remained statistically unaltered on lead exposure. Brain catalase activity, on the other hand, increased significantly. Administration of LA was effective in reducing these alterations in the brain, however, the best effects were achieved in animals co-administered LA and one of the thiol chelators. The results point to a significant beneficial role of LA in the recovery of altered biochemical variables both during monotherapy and when given in combination with succimer. It however, showed no chelating properties in decreasing lead burden from blood, liver and kidneys except for a significantly more pronounced decrease in brain lead concentration in animals administered LA plus thiol chelators, compared to the effects of chelating agents alone. This is an interesting and notable observation, which requires further exploration. The results thus provide evidence of an encouraging role of LA when given in combination with a thiol chelator in the therapeutic intervention of lead poisoning, particularly in reducing the oxidative stress and brain lead concentration.

Kinetics of aluminum-induced inhibition of δ-aminolevulinic acid dehydratase In vitro

Anemia, one consequence of aluminum toxicity, may be due to inhibition of enzymes in the heme biosynthetic pathway. In this study, the in vitro effect of aluminum on rat liver and erythrocyte δ-aminolevulinic acid dehydratase (8-ALA dehydratase), an enzyme that is sensitive to a number of metal ions, was investigated. The presence of 1–10 μM AlCl 3 caused a concentration-dependent inhibition of liver δ-ALA dehydratase activity. The K i for AlCl 3-induced inhibition of δ-ALA dehydratase was 4.1 μM, and 10 μM AlCl 3 virtually abolished δ-ALA dehydratase activity (99% inhibition). Erythrocyte δ-ALA dehydratase was also inhibited by similar concentrations of AlCl 3 and displayed a K i of 1.1 μM. AlCl 3 (5 μM) decreased the V max by 50% but did not change the K m , suggestive of reversible, noncompetitive inhibition. Sodium citrate (50 μM) when added with AlCl 3 completely restored δ-ALA dehydratase activity to basal levels. Thus, disruption of δ-ALA dehydratase occurred at low micromolar levels of AlClP 3 in vitro, which may help to explain abnormalities in the heme pathway in cases of aluminum poisoning.

Effect of alcohol on δ-aminolevulinic acid dehydratase and porphyrin metabolism in man

The influence of alcohol on porphyrin metabolism was investigated in 6 healthy non-alcoholics and 19 patients with chronic alcohol abuse. In the healthy subjects, blood and urine samples were obtained before and after acute alcohol exposure, whereas in the chronic alcoholics only one examination was performed. In both groups a significant inhibition of δ-aminolevulinic acid dehydratase was demonstrated. The activity was partially restored in vitro by addition of zinc ions or dithiothreitol. A combination of both activators produced reactivation to normal levels. Coproporphyrinuria was more prominent in chronic alcoholics (373 nmol/24 h on average, upper norm 119 nmol/24 h) compared to non-alcoholics (140 nmol/24 h). Urinary porphobilinogen and δ-amino-levulinic acid were normal except for a moderately increased δ-aminolevulinic acid in four healthy individuals. In conclusion, alcohol causes reversible inhibition of δ-aminolevulinic acid dehydratase. The metabolic changes reflect both an inhibition of δ-aminolevulinic acid dehydratase and coproporphyrinogen oxidase; a simultaneous, moderate induction of hepatic δ-aminolevulinic acid synthase is suggested. Erythrocyte δ-aminolevulinic acid dehydratase activity could serve as a sensitive indicator for both acute and chronic alcohol consumption even better than Coproporphyrinuria.

Interaction of tin and zinc in some processes of heme biosynthesis in rabbits

Female rabbits (2.5–3.0 kg) were divided into the five groups. Group I (control) received ip 0.9% NaCl, every day for 5 days; group II received ip SnCl 2 (dose of 2 mg Sn/kg) every day for 5 days; group III was given single sc injection of ZnSO 4 (dose of 50 mg Zn/kg); group IV, a single sc injection of ZnSO 4 followed by a single ip injection of SnCl 2 on the next day; and group V received a single ip injection of SnCl 2 followed next day by a single sc injection of ZnSO 4. Eighteen hours after the first dose of SnCl 2 and inhibition of δ-aminolevulinic acid dehydratase (ALA-D) by 60% was found in the blood of group II, while increases of urinary excretion of coproporphyrin (CP-U) and δ-aminolevulinic acid (ALA-U) with respect to the control group took place not earlier than after the fourth dose of SnCl 2 (correlation coefficients, respectively: ALA-D/ALA-U r = −0.91; CP-U/ALA-D r = −0.70; CP-U/ALA-U r = 0.81). The single dose of ZnSO 4 resulted in an increase of ALA-D by about 60% while administration of SnCl 2 to the animals in group IV decreased this value down to physiologic levels. Then an increased tendency was noted again for the activity of this enzyme. Administration of SnCl 2 and ZnSO 4 (group V) resulted in a decrease of ALA-D activity and in an increase in CP-U.

Inhibition of δ-aminolevulinic acid dehydratase by trichloroethylene

Male Wistar rats (8 animals/group; 180–200 g) were exposed continuously to trichloroethylene (TRI) for 48 or 240 h or methylchloroform (1,1,1-trichloroethane: MC for 48 h at 50, 400 and 800 ppm. The inhibition of δ-aminolevulinic acid dehydratase (ALA-D) was examined in liver, blood and bone marrow of naive and phenobarbital pretreated animals exposed to TRI. A clear cut dose-effect relationship between the exposure concentration or duration of exposure and the inhibition of ALA-D activity was seen for rats exposed to TRI. In addition to this finding, significant interaction between TRI exposure and phenobarbital treatment was observed in the inhibition of ALA-D in liver and blood. MC did not produce inhibition. Trichloroacetic acid and trichloroethanol failed to inhibit the ALA-D activity in vitro. It seems that a metabolite(s) of TRI other than the above 2 substances may play a role in the inhibition of ALA-D. The inhibition of ALA-D (38% or 48% of the control in liver or in blood, respectively) observed after the 240 h exposure at 400 ppm to TRI was accompanied by the significant elevation of δ-aminolevulinic acid synthase (186% of the control) in liver and the increase in excretion of δ-aminolevulinic acid in urine (142% of the control). This occurred without an apparent weight loss, liver injury or hematological changes.

Selective inhibition of δ-aminolevulinic acid dehydratase by indium chloride in rat kidney: Biochemical and ultrastructural studies

Indium chloride, a nephrotoxic and porphyrogenic chemical, exerts a tissue-specific inhibition of δ-aminolevulinic acid (ALA) dehydratase in rat kidney. This effect appears to be related to the preferential accumulation of indium by the kidney proximal tubule cell and to the selective localization of indium within the cytosolic fraction of that cell type, as assessed by electron microscopy. The reversal of indium-induced inhibition of renal ALA dehydratase both in vivo and in vitro by zinc suggests that indium acts by binding with sulfhydryl groups of the enzyme. The synergistic effects of lead and indium with respect to inhibition of renal ALA dehydratase suggest that exposure to weak sulfhydryl inhibitors such as the group III metals may enhance the renal toxicity of other metals which may not in themselves exert overt toxic effects in that organ.

Antagonistic Effects of Zinc and Aluminum on Lead Inhibition of δ-Aminolevulinic Acid Dehydratase

Antagonistic Effects of Zinc and Aluminum on Lead Inhibition of δ-Aminolevulinic Acid Dehydratase

Hemin feedback inhibition at reticulocyte δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydratase

Addition of hemin (5–200 μM) to a rabbit reticulocyte iron-free incubation medium, resulted in a progressive inhibition of heme synthesis as measured by incorporation of ( 14C)-glycine. In contrast when ( 14C) δ-aminolevulinic acid incorporation into heme was studied, significant inhibition below that of the ( 14C)-glycine control only occurred with hemin concentrations greater than 100 μM. Hemin progressively inhibited cellular and mitochondrial δ-aminolevulinic acid synthetase activity, as well as cellular δ-aminolevulinic acid dehydratase activity. The results indicated that elevated levels of hemin initially control heme synthesis by feedback inhibition at the rate-limiting enzyme of heme synthesis, δ-aminolevulinic acid synthetase. Hemin inhibition of δ-aminolevulinic acid dehydratase is only significant for the entrire heme synthetic pathway when greater than one-third of this enzyme's activity is inhibited.

Effects of lead on the induction of hepatic microsomal enzymes by phenobarbital and 3,4-benzpyrene

A single ip injection of lead acetate (65 mg/kg) prolonged the duration of zoxazolamine paralysis in Sprague-Dawley rats and prevented the stimulation of zoxazolamine metabolism normally induced by phenobarbital. Lead also delayed the phenobarbital induction of hepatic microsomal cytochrome P-450 at 12 and 24 hr after treatment. However, the inhibitory effect of lead had disappeared and cytochrome P-450 concentrations were markedly elevated at 48 hr. Lead also diminished hepatic microsomal cytochrome P-448, normally resulting from benzyprene treatment. Neither lead, phenobarbital, benzyprene nor their combination had a significant effect on hepatic cytochrome b 5 values. Lead, in vitro, inhibited the hepatic microsomal NADPH-cytochrome c and P-450 reductase activity in a dose-dependent manner. In vivo, significant inhibition of NADPH-cytochrome c and P-450 reductase activity was observed after the injection of lead acetate. Significant inhibition coincides with the time when lead concentration in the hepatic microsomes was maximal. The inhibition of δ-aminolevulinic acid dehydratase (ALAD) activity at several blood lead concentrations was confirmed both in vivo and in vitro. This relationship between blood lead concentrations and ALAD activity was not altered by pretreatment with phenobarbital or benzpyrene.

Studies on δ-Aminolevulinic Acid Synthetase Activity in Bone Marrow in Experimental Lead Poisoned Rats

Urinary increase of δ-aminolevulinic acid and coproporphyrin as well as basophilic stippled cells in the peripheral blood are the characteristic signs of lead poisoning. The biochemical lesion of porphyrin biosynthesis in lead poisoning is the partial inhibition of δ-aminolevulinic acid dehydratase, but whether or not δ-aminolevulinic acid synthetase is induced (stimulated) or not by lead is still unknown. This paper reports that δ-aminolevulinic synthetase activity in bone marrow in lead poisoned rats was 6.8 times higher than that of normal rats. Activity was found to increase within three or four days after lead administration and in parallel with the urinary increase of δ-aminolevulinic acid.The levels of urinary δ-aminolevulinic acid and coproporphyrin in lead poisoned rats decreased markedly following the administration of mitomycin c. This phenomenon were also observed in the case of hepatic porphyria caused by diethyl-1, 4-dihydro-2, 4, 6-trimethylpyridine-3, 5-dicarboxylate poisoned rats when treated with mitomycin c.These results strongly suggest that δ-aminolevulinic acid synthetase activity is induced either directly or indirectly by lead. Excessive δ-aminolevulinic acid thus formed seems to overcome the partial blocking of δ-aminolevulinic acid dehydratase resulting in the increased formation of coproporphyrin in lead poisoning.

The relationship between inhibition of δ-aminolevulinic acid dehydratase by lead and lead mobilization by ethylenediamine tetraacetate (EDTA)

The effect of ethylenediamine tetraacetate (EDTA) on the activity of δ-aminolevulinic acid dehydratase (ALAD) and on the excretion of lead in the urine of lead-intoxicated rats is reported. Rats were given lead in their drinking water at 0.5 and 1% for varying periods of time or as single iv doses of 20–30 mg/kg. All the animals experienced a pronounced reduction in weight gains. Lead caused inhibition of ALAD in both blood and liver. Inhibition was greater in blood than in liver. Although EDTA itself caused slight inhibition of ALAD in rats not given lead, EDTA treatment of rats given lead resulted in rapid reactivation of the ALAD in liver. By contrast, EDTA treatment had little effect on lead-induced ALAD inhibition in blood. EDTA-induced lead excretion in the urine was inversely proportional to the liver ALAD activity. The correlation of the concentration of lead in the liver before treatment with ALAD activity was equally good. The results are discussed in regard to the value of the EDTA lead-mobilization test as a measure of the toxic fraction of the body burden of lead.

Inhibition of δ-aminolevulinic acid dehydratase of Rhodopseudomonas spheroides by urea, guanidine and methyl derivatives of urea

A purified preparation of δ-aminolevulinic acid dehydratase (EC 4.2.1.24) of R. spheroides is inhibited by low concentrations of urea, guanidine, and methyl derivatives of urea. The inhibition by urea and guanidine has been found to be competitive in nature—a fact that has been supported by mixed inhibition study indicating the competition for the same site as that of the substrate. The dissociation of the enzyme by urea into subunits and its prevention from dissociation by the substrate point to their opposing effects on the protein conformation rather than their competition for identical sites of action.