Inhibition Of ALAD Research Articles

Protective role of hydroxy citric acid (HCA) against lead induced toxicity in albino wistar rats

The present study was designed to evaluate the role of herbal active constituent hydroxycitric acid obtained from Garcinia cambogia and Hibiscus subdariffa in heavy metal especially lead poisoning. The Lead acetate at 500ppm was used as an inducing agent in the present study. Sodium acetate (500ppm) used as control group is used as a baseline measure. The HCA at 100 mg/kg and 200mg/kg were used in the treatment of lead induced toxicity in rats. At the end of 28 days study period the blood levels of alpha ALAD activity was estimated in all the treatment groups. Lead inhibits the ALAD is more profound and its inhibition has been used clinically to gauge the degree of lead poisoning. Inhibition of ALAD results in the accumulation of aminolaevulinic acid, detectable in the plasma and urine even at blood lead levels of less than 10 µg/dl. It results that HCA showed significant elevated levels of ALAD activity, which was reduced by the lead intoxication. HCA showed significant reduction of liver enzymes (SGOT, SGPT and ALP). The markers like protein, bilirubin and creatinine were also found to be elevated in lead intoxicated rats and was found to be decreased significantly in a concentration dependent manner.

Integrative analysis of multiomics data identifies selenium-related gene ALAD associating with keshan disease.

Keshan disease is an endemic fatal dilated cardiomyopathy that can cause heart enlargement, heart failure, and cardiogenic death. Selenium deficiency is considered to be the main cause of Keshan disease. However, the molecular mechanism underlying Keshan disease remains unclear. Our whole-exome sequencing from 68 patients with Keshan disease and 100 controls found 199 candidate genes by gene-level burden tests. Interestingly, using multiomics data, the selenium-related gene ALAD (δ-aminolevulinic acid dehydratase) was the only candidate causative gene identified by three different analysis approaches. Based on single-cell transcriptome data, ALAD was highly expressed in cardiomyocytes and double mutations of human ALAD dramatically reduced its enzyme activity in vitro compared to negative control. Functional analysis of ALAD inhibition in mice resulted in a Keshan phenotype with left ventricular enlargement and cardiac dysfunction, whereas administration of sodium selenite markedly reversed the changes caused by ALAD inhibition. In addition, sodium selenite reversed Keshan phenotypes by affecting energy metabolism and mitochondrial function in mice as shown by the transcriptomic and proteomic data and the ultrastructure of cardiac myocytes. Our findings are the first to demonstrate that the selenium-related gene ALAD is essential for cardiac function by maintaining normal mitochondrial activity, providing strong molecular evidence supporting the hypothesis of selenium deficiency in Keshan disease. These results identified ALAD as a novel target for therapeutic intervention in Keshan disease and Keshan disease-related dilated cardiomyopathy.

ALAD Inhibition by Porphobilinogen Rationalizes the Accumulation of δ-Aminolevulinate in Acute Porphyrias.

Patients with major forms of acute hepatic porphyria present acute neurological attacks with overproduction of porphobilinogen (PBG) and δ-aminolevulinic acid (ALA). Even if ALA is considered the most likely agent inducing the acute symptoms, the mechanism of its accumulation has not been experimentally demonstrated. In the most frequent form, acute intermittent porphyria (AIP), inherited gene mutations induce a deficiency in PBG deaminase; thus, accumulation of the substrate PBG is biochemically obligated but not that of ALA. A similar scenario is observed in other forms of acute hepatic porphyria (i.e., porphyria variegate, VP) in which PBG deaminase is inhibited by metabolic intermediates. Here, we have investigated the molecular basis of δ-aminolevulinate accumulation using in vitro fluxomics monitored by NMR spectroscopy and other biophysical techniques. Our results show that porphobilinogen, the natural product of δ-aminolevulinate deaminase, effectively inhibits its anabolic enzyme at abnormally low concentrations. Structurally, this high affinity can be explained by the interactions that porphobilinogen generates with the active site, most of them shared with the substrate. Enzymatically, our flux analysis of an altered heme pathway demonstrates that a minimum accumulation of porphobilinogen will immediately trigger the accumulation of δ-aminolevulinate, a long-lasting observation in patients suffering from acute porphyrias.

Zingerone prevents lead-induced toxicity in liver and kidney tissues by regulating the oxidative damage in Wistar rats.

Among the heavy metal poisonings, lead is considered as a major toxic metal causing hematological, neurological, immunological, hepatic, and renal dysfunctions. Lead causes inhibition of ALAD leading to the ALA accumulation inside the cells. Lead also leads to disruption of the anti-oxidative enzyme system, organ function, and lipid membranes of the cell causing oxidative stress. Zingerone, a phenolic alkanone, is an active edible ingredient present in the ginger that possess varied pharmacological properties. The aim of our study was to evaluate the protective effect of zingerone in lead-induced toxicity in wistar rats. ALAD concentration was improved in kidney and liver tissues treated with zingerone. Protective effect of zingerone was observed in terms of significant improvement in kidney and liver histology, anti-oxidant enzyme activity (CAT, SOD, GPx, and GR), organ function parameters, lipid profile, and decreased level of LPO. Therefore, zingerone pretreatment can be a promising agent for alleviation of lead-induced oxidative damage in cells. PRACTICAL APPLICATIONS: Published reports have revealed that consumption of certain bioactive nutrients for example, flavonoids, mineral elements, and vitamins can offer defense from the environmental lead contamination. Zingerone is a strong anti-oxidant, with very less side effects and has exceptional property of scavenging free radicals, hence reducing the oxidative stresses. This fundamental property of zingerone can alone help in countering the heavy metal toxicity. Different groups have published reported numerous properties of zingerone but as per our understanding till date no study about alleviation of lead toxicity by zingerone in animal model has been undertaken. Hence, we conducted this research to explore the preventive effect of zingerone in lead induced kidney and liver toxicity. The outcome of our study shows potent anti-oxidant effect and ALAD modulatory property of zingerone which makes it suitable edible candidate for use in countering lead toxicity.

Experimental manipulation of dietary lead levels in great tit nestlings: limited effects on growth, physiology and survival

We manipulated dietary lead (Pb) levels of nestlings in wild populations of the great tit (Parus major L) to find out if environmentally relevant Pb levels would affect some physiological biomarkers (haematocrit [HT], fecal corticosterone metabolites [CORT], heat shock proteins [HSPs], erythrocyte delta-aminolevulinic acid dehydratase activity [ALAd]), growth (body mass, wing length), phenotype (plumage coloration) or survival of nestlings. The responses to three experimental manipulation (control, low and high: 0, 1 and 4 μg/g body mass/day) are compared with those in a P. major population breeding in the vicinity of a heavy metal source, a copper smelter. Our Pb supplementation was successful in raising the fecal concentrations to the levels found in polluted environments (high: 8.0 μg/g d.w.). Despite relatively high range of exposure levels we found only few effects on growth rates or physiology. The lack of blood ALAd inhibition suggests that the circulating Pb levels were generally below the toxic level despite that marked accumulation of Pb in femur (high: 27.8 μg/g d.w.) was observed. Instead, birds in the metal polluted environment around the smelter showed decreased growth rates, lower HT, higher CORT, less colorful plumage and lower survival probabilities than any of the Pb treated groups. These effects are likely related to decreased food quality/quantity for these insectivorous birds at the smelter site. In general, the responses of nestlings to metal exposure and/or associated resource limitation were not gender specific. One of the stress proteins (HSP60), however, was more strongly induced in Pb exposed males and further studies are needed to explore if this was due to higher accumulation of Pb or higher sensitivity of males. In all, our results emphasize the importance of secondary pollution effects (e.g. via food chain disruption) on reproductive output of birds.

Lead-Binding Proteins: A Review

Lead-binding proteins are a series of low molecular weight proteins, analogous to metallothionein, which segregate lead in a nontoxic form in several organs (kidney, brain, lung, liver, erythrocyte). Whether the lead-binding proteins in every organ are identical or different remains to be determined. In the erythrocyte, delta-aminolevulinic acid dehydratase (ALAD) isoforms have commanded the greatest attention as proteins and enzymes that are both inhibitable and inducible by lead. ALAD-2, although it binds lead to a greater degree than ALAD-1, appears to bind lead in a less toxic form. What may be of greater significance is that a low molecular weight lead-binding protein, approximately 10 kDa, appears in the erythrocyte once blood lead exceeds 39 μg/dL and eventually surpasses the lead-binding capacity of ALAD. In brain and kidney of environmentally exposed humans and animals, a cytoplasmic lead-binding protein has been identified as thymosin β4, a 5 kDa protein. In kidney, but not brain, another lead-binding protein has been identified as acyl-CoA binding protein, a 9 kDa protein. Each of these proteins, when coincubated with liver ALAD and titrated with lead, diminishes the inhibition of ALAD by lead, verifying their ability to segregate lead in a nontoxic form.

Possíveis efeitos do cobre sanguíneo sobre parâmetros hematológicos em idosas

INTRODUÇÃO: O cobre é um elemento traço essencial e sua homeostase é importante, principalmente em idosos, uma vez que seu metabolismo está associado a doenças neurodegenerativas e distúrbios na eritropoiese, entre outros. OBJETIVO: O presente estudo avaliou a associação entre cupremia, parâmetros hematológicos e estresse oxidativo. MATERIAIS E MÉTODOS: Amostras de sangue de 39 mulheres idosas (grupo de estudo) e de 20 indivíduos adultos saudáveis (grupo-controle) foram coletadas. As concentrações de cobre sérico foram quantificadas por espectrometria de massa por plasma indutivamente acoplado (ICP-MS), a atividade e o índice de reativação da enzima δ-aminolevulinato desidratase (ALA-D) foram determinados por espectrofotometria e parâmetros sanguíneos foram analisados em sistema automatizado. Os resultados foram expressos em média ± desvio padrão (DP). RESULTADOS: As concentrações de cobre, parâmetros hematológicos e índice de reativação da ALA-D para ambos os grupos encontraram-se dentro dos valores de referência. Porém, a atividade da ALA-D (11,47 ± 2,81 U.L-1) foi significativamente inferior no grupo de estudo em comparação com o grupo-controle. Correlações de Spearman (observadas somente nas mulheres idosas) entre as concentrações de cobre versus hemoglobina, hematócrito e atividade da ALA-D foram -0,384; -0,408 e -0,395, respectivamente (p < 0,05). No entanto, o índice de reativação da ALA não apresentou correlação com a cupremia. DISCUSSÃO E CONCLUSÃO: Os resultados mostraram que o cobre, mesmo estando nos limites considerados valores de referência, pode estar envolvido na inibição da ALA-D, o que pode alterar parâmetros hematológicos, como a síntese de hemoglobina. Neste sentido, sugere-se que os níveis de referência para o cobre em idosos sejam reavaliados.

Comparison of ALAD activities of Citrobacter and Pseudomonas strains and their usage as biomarker for Pb contamination

Delta-aminolevulinate dehydratase (ALAD) activity has been used in prokaryotes and eukaryotes as a biomarker for environmental lead (Pb) exposure and toxicity. Microorganisms are sensitive indicators of toxicity at the fundamental level of ecological organization, but bacterial biomarker studies are focused on the Pseudomonas strains in Group I and E coli. The objectives of the present work were to determine if Burkholderia gladioli belonging to group II, due to its 16SrRNA similarity, can be used as biomarker in metal contamination and compare its possible usage with Pseudomonas aeruginosa and Citrobacter freundii (previously known as Esherichia freundii) and Bacterium freundii which are classified in Group I. In this study, ALAD activity in an environmental strains of Burkholderia gladioli, Pseudomonas aeruginosa, Citrobacter freundii were investigated to evaluate potential inhibition by Pb and other toxic metals. When the ALAD activity of Burkholderia gladioli was tested, Co and Pb decreased activity by 27 and 71%, respectively. In addition to these findings, Zn increased the activity up to 26%. These effects were found to be statistically meaningful (p < 0.05). It was determined that the increase of lead concentration inhibites the ALAD activity at each of the three strains. There was a statistically significant dose-response relationship between ALAD activity in cells of Burkholderia gladioli and Pb (Pearson correlation coefficent = -0.665; r(2) = 0.665, and p < 0.001). The strongest ALAD inhibition which was measured was 90% at Burkholderia gladioli when protein extracts were incubated with 750 muM of Pb. The relationship between Pb and ALAD activity was statistically described by [ALAD Activity] = 0.476-0.000597 x [Pb]. According to the obtained results, we suggest that the ALAD of Burkholderia gladioli can be used as a biomarker for lead contamination in the environment.

Human erythrocyte δ-aminolevulinate dehydratase activity and oxidative stress in hemodialysis patients

Background: Oxidative stress is a complicating factor in chronic renal failure, especially in hemodialysis (HD) patients. Also, aluminum intoxication may occur during hemodialysis treatment. Aluminum has been shown to inhibit the sulfhydryl-containing enzyme δ-aminolevulinate dehydratase (ALA-D). Thus, the involvement of –SH oxidation in ALA-D inhibition and its relationship with serum Al levels and lipid peroxidation in HD patients were evaluated. Methods: Blood ALA-D activity, plasma thiobarbituric acid reactive substances (TBARS), and serum aluminum levels were measured in HD patients ( n = 37) and healthy controls ( n = 20). Results: TBARS and Al levels were higher in HD patients than in controls ( p < 0.01), while ALA-D activity was lower ( p < 0.05). The sulfhydryl-reducing agent dithiothreitol (DTT) reactivated ALA-D of HD patients, but activity was still lower than that of controls. ALA-D activity was negatively correlated with TBARS ( r = − 0.63, p < 0.01) and aluminum levels ( r = − 0.31, p < 0.05). Conclusions: Reduced ALA-D activity in HD patients was found to be related to the oxidation of –SH groups essential for enzyme activity. Our results suggest that increased oxidative stress may have contributed to enzyme inhibition in HD patients.

Oxidative stress and δ-ALA-D activity in chronic renal failure patients

In this paper, we studied the influence of uremia and hemodialysis on oxidative parameters and δ-aminolevulinic acid dehydratase (δ-ALA-D) activity in control subjects, patients with chronic renal failure (CRF) on hemodialysis treatment (HD) and in patients not undergoing hemodialysis (ND). An increased lipid peroxidation was observed in the serum of HD and ND patients, as measured by the MDA serum levels. However, the level of MDA from erythrocytes was only elevated in HD patients. Blood catalase activity was increased in HD and ND groups. This study also showed a decreased activity of blood δ-aminolevulinic acid dehydratase (δ-ALA-D) in both groups of patients. This study demonstrated a positive correlation between ALA-D activity and hemoglobin, suggesting that inhibition of this enzyme might enhance anemia in CRF. A negative correlation was found between the alteration in δ-ALA-D activity and oxidative stress, which may indicate that the inhibition of ALA-D can be used as an index of oxidative stress.

Beneficial role of monoesters of meso-2,3-dimercaptosuccinic acid in the mobilization of lead and recovery of tissue oxidative injury in rats

We investigated the therapeutic efficacy of meso-2,3-dimercaptosuccinic acid (DMSA) and two of its analogues, monomethyl dimercaptosuccinic acid (MmDMSA) and mono-cyclohexyl dimercaptosuccinic acid (MchDMSA) in reducing lead concentration in blood and soft tissues, and in recovering lead induced oxidative stress in rats. Male wistar rats were exposed to lead acetate in drinking water for 20 weeks, followed by 5 days of oral treatment with DMSA (100 mg/kg, oral, once daily), MmDMSA or MchDMSA (50 and 100 mg/kg). Biochemical variables indicative of oxidative stress along with lead, zinc and copper concentration were evaluated in blood and other soft tissues. Exposure to lead caused a significant decrease in blood δ-aminolevulinic acid dehydratase (ALAD) activity and glutathione (GSH) level. These changes were accompanied by inhibition of kidney ALAD and an increase in δ-aminolevulinic acid synthatase (ALAS) activity in liver and kidneys. Also seen were a pronounced depletion of brain GSH, glutathione peroxidase (GPx), glutathione- S-transferase (GST) and decreased superoxide dismutase (SOD) activity and an increase in thiobarbituric acid reactive substances (TBARS) and reactive oxygen species (ROS) levels. These biochemical changes were correlated with an increased uptake of lead in blood and soft tissues. Blood and kidneys zinc concentration decreased significantly following lead exposure while, copper concentration remained unchanged. No effect of chelation on hepatic zinc concentration was noted, only liver copper concentration showed significant depletion on treatment with DMSA and MmDMSA (100 mg/kg). Treatment with DMSA, MmDMSA and MchDMSA provided significant recovery in altered biochemical variables and brain DNA damage besides significant depletion of tissue lead burden. Among the chelating agents used, MchDMSA and MmDMSA provided better recovery in altered biochemical variables and depletion of lead concentration in tissues compared to DMSA. The above results suggest DMSA monoesters to be a better treatment option than DMSA in eliciting recovery to the altered biochemical variables and in the depletion of body lead burden.

Effects of mercury and selenite on $delta;-aminolevulinate dehydratase activity and on selected oxidative stress parameters in rats*1

The present study evaluates the effects of Na 2 SeO 3 and HgCl 2 on kidney and liver of adult rats. In vivo, HgCl 2 (17 μmol/kg, sc ) reduced ascorbic acid levels in liver (∼15%), whereas in kidney it reduced ALA-D activity (∼60%) and ascorbic acid levels (∼35%) and increased TBARS content (∼50%). Na 2 SeO 3 (17 μmol/kg, sc ) exposure increased the content of nonprotein thiol groups in liver (35–60%) and kidney (∼50–160%), partially prevented mercury-induced ALA-D inhibition in kidney, and completely prevented a mercury-induced increase of TBARS content and decrease of ascorbic acid levels in kidney. In vitro, HgCl 2 and Na 2 SeO 3 inhibited renal and hepatic ALA-D, while HgCl 2 increased TBARS in renal and hepatic tissue preparations. Na 2 SeO 3 increased the rate of glutathione oxidation in vitro. Results indicated that Na 2 SeO 3 protected against HgCl 2 effects in vivo (prevention of mercury interaction with thiol groups and of mercury-induced oxidative damage). In vitro, Na 2 SeO 3 did not prevent mercury effects, but potentiated ALA-D inhibition by mercury, probably due to its ability to oxidize thiol groups.

Effects of mercury and selenite on δ-aminolevulinate dehydratase activity and on selected oxidative stress parameters in rats

The present study evaluates the effects of Na 2SeO 3 and HgCl 2 on kidney and liver of adult rats. In vivo, HgCl 2 (17 μmol/kg, sc) reduced ascorbic acid levels in liver (∼15%), whereas in kidney it reduced ALA-D activity (∼60%) and ascorbic acid levels (∼35%) and increased TBARS content (∼50%). Na 2SeO 3 (17 μmol/kg, sc) exposure increased the content of nonprotein thiol groups in liver (35–60%) and kidney (∼50–160%), partially prevented mercury-induced ALA-D inhibition in kidney, and completely prevented a mercury-induced increase of TBARS content and decrease of ascorbic acid levels in kidney. In vitro, HgCl 2 and Na 2SeO 3 inhibited renal and hepatic ALA-D, while HgCl 2 increased TBARS in renal and hepatic tissue preparations. Na 2SeO 3 increased the rate of glutathione oxidation in vitro. Results indicated that Na 2SeO 3 protected against HgCl 2 effects in vivo (prevention of mercury interaction with thiol groups and of mercury-induced oxidative damage). In vitro, Na 2SeO 3 did not prevent mercury effects, but potentiated ALA-D inhibition by mercury, probably due to its ability to oxidize thiol groups.

Effect of Ni2+ on Early Stages of Chlorophyll Biosynthesis and Pheophytinization in Euglena gracilis

The effect of Ni2+ on the early stages of chlorophyll biosynthesis and pheophytinization in Euglena gracilis cells was studied. Incubation of the cells with 10–4 M Ni2+ for 7 days resulted in a higher chlorophyll content, enhanced production of 5-aminolevulinic acid (ALA), and in increased activity of 5-aminolevuluinic acid dehydratase (EC 4.2.1.24, ALAD), as compared to the control cells incubated without Ni2+. At a higher concentration (10–3 M), Ni2+ markedly inhibited chlorophyll accumulation and ALAD activity, as compared to the control cells. At this concentration, Ni2+ also inhibited heme biosynthesis and strongly stimulated ALA production. It seems likely that, by affecting heme synthesis, Ni2+ increases the activity of the ALA production system. However, the suppression of subsequent stages of ALA conversion to chlorophyll, in particular ALAD inhibition, ultimately resulted in almost complete inhibition of chlorophyll biosynthesis. In addition to cessation of de novo chlorophyll synthesis in the presence of Ni2+ (10–3 M) in Euglena cells, the existing chlorophyll was converted into pheophytin and almost completely degraded. We suppose that the Ni2+-induced pheophytinization is caused by an acidic shift of intracellular pH related to an impairment of cell membrane permeability by Ni2+ cations.

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with substrate and three inhibitors

The structures of 5-aminolaevulinic acid dehydratase (ALAD) complexed with substrate (5-aminolaevulinic acid) and three inhibitors: laevulinic acid, succinylacetone and 4-keto-5-aminolaevulinic acid, have been solved at high resolution. The ligands all bind by forming a covalent link with Lys263 at the active site. The structures define the interactions made by one of the two substrate moieties that bind to the enzyme during catalysis. All of the inhibitors induce a significant ordering of the flap covering the active site. Succinylacetone appears to be unique by inducing a number of conformational changes in loops covering the active site, which may be important for understanding the co-operative properties of ALAD enzymes. Succinylacetone is produced in large amounts by patients suffering from the hereditary disease type I tyrosinaemia and its potent inhibition of ALAD also has implications for the pathology of this disease. The most intriguing result is that obtained with 4-keto-5-amino-hexanoic acid, which seems to form a stable carbinolamine intermediate with Lys263. It appears that we have defined the structure of an intermediate of Schiff base formation that the substrate forms upon binding to the P-site of the enzyme.

Heme biosynthesis and oogenesis in the blood-sucking bug, Rhodnius prolixus

We have previously shown that the pathway of porphyrin synthesis operates in the blood feeding triatomine bug Rhodnius prolixus but not in the cattle tick Boophilus microplus. In the present paper we studied the correlation between heme synthesis and egg development in Rhodnius. There is a sharp increase heme biosynthetic capability in the fat body (160%) and in the ovaries (360%) in response to a blood meal, as evaluated from the activity of the enzyme δ-aminolevulinate dehydratase (EC 4.2.1.24). The in vivo inhibition of ALA-D by succinyl acetone results in a dose dependent decrease of oviposition. Oviposition is recovered when porphobilinogen, the product of the impaired reaction, is added to the succinyl acetone enriched blood. Taken together, these results show that heme biosynthesis is a fundamental event to vitellogenic females. The demand for heme in this metabolic juncture cannot be supplied by the heme eventually absorbed during blood digestion and associated with Rhodnius heme binding protein (RHBP), which is then incorporated into growing oocytes. Inhibition of heme biosynthesis results in lower levels of RHBP in the hemolymph, suggesting that the synthesis of this protein is controlled by heme availability.

Microbial δ-aminolevulinate dehydratase as a biosensor of lead bioavailability in contaminated environments

Delta-aminolevulinate dehydratase (ALAD) is a phylogenetically conserved enzyme that is responsible for the synthesis of porphobilinogen in a key step involving the production of heme. ALAD is a metalloprotein that requires magnesium or zinc for its activity, depending on the species. In humans, ALAD activity is zinc-dependent, and has been used extensively as a biomarker for lead (Pb) exposure and toxicity. ALAD activity in other multicellular eukaryotes has been used in ecotoxicology as a biomarker for environmental lead pollution. Because microorganisms are sensitive indicators of toxicity at the fundamental level of ecological organization, we hypothesized that bacterial ALAD can serve as a reliable biomarker for lead bioavailability in contaminated environments. In this study, ALAD activity in an environmental strain of Pseudomonas putida was investigated to evaluate potential inhibition by Pb and other toxic metals. There was a statistically significant dose–response relationship between ALAD activity in cells of P. putida, ATCC 700097 and [Pb] (Pearson correlation coefficient=−0.985; r 2=0.97, and P<0.001). The highest level of inhibition of ALAD activity was approximately 74% of the normal level when cells were incubated with [Pb 2+]>500 μM. The relationship between Pb and ALAD activity was statistically described by log[ Pb]=3.68−1.41[ ALAD Activity ]. In protein extracts of P. putida, ALAD activity was reduced by up to 85% in response to 500 μM of Pb. A higher concentration of Pb was needed to produce a comparable level of ALAD inhibition in P. putida cells seeded into natural freshwater, suggesting that Pb was not completely bioavailable in the water samples. In contrast to the findings with P. putida, the ALAD activity in a known metal-resistant P. aeruginosa PU21 (Rip64) was not sensitive to Pb exposure. Therefore, the sensitivity of ALAD to Pb in complex heterogenous ecosystems depends on the molecular diversity of ALAD in predominant species, and on the bioavailability of Pb.

Lead exposure in passerines inhabiting lead‐contaminated floodplains in the Coeur D'Alene River Basin, Idaho, USA

AbstractBlood collected from song sparrows (Melospiza melodia) and American robins (Turdus migratorius) captured with mist nets in a lead‐contaminated (assessment) area and nearby uncontaminated (reference) areas within the Coeur d'Alene Basin in northern Idaho was analyzed for δ‐aminolevulinic acid dehydratase activity (ALAD) and hematocrit levels, and livers were analyzed for lead. Mean ALAD inhibition in the assessment area was 51% in song sparrows and 75% in American robins. The proportion of the sampled population with ALAD inhibition &gt;50% was calculated to be 43% for song sparrows and 83% for American robins. Assessment area hematocrit values for song sparrows (x = 39.9) and American robins (x = 39.5) were lower than in reference areas (x = 42.4 for song sparrows and 40.2 for American robins); however, differences were not statistically significant (p &gt; 0.05). Significantly higher levels of lead (wet weight) were found in livers from song sparrows captured on the assessment area (x = 1.93 ppm) than on reference areas (x = 0.10 ppm) (p = 0.0079). Study results indicate that 43% (95% confidence interval [CI] = 12.9‐77.5%) of the song sparrows and 83% (95% CI = 41.8‐99.2%) of the American robins inhabiting the floodplain along the Coeur d'Alene River in the assessment area are being exposed to lead at levels sufficient to inhibit ALAD by &gt; 50%. Variability in lead exposure indicators was attributed to high variability in environmental lead concentrations in the Coeur d'Alene River Basin.

Delta-Aminolevulinate dehydratase inhibition by 2,3-dimercaptopropanol is mediated by chelation of zinc from a site involved in maintaining cysteinyl residues in a reduced state.

The mechanisms underlying mouse delta-aminolevulinate dehydratase (ALA-D) inhibition by a chelating agent used in the treatment of heavy metal poisoning, 2,3-dimercaptopropanol (British Anti-Lewisite), were investigated. ALA-D inhibition by 2,3-dimercaptopropanol was totally reversed by 25-100 microM Zn2+, indicating that inhibition was due to chelation of zinc by 2,3-dimercaptopropanol. Our data suggested that zinc bound to a labile site (displaced by 25-40 microM EDTA or 500 microM 2,3-dimercaptopropanol) is involved in maintaining the sulfhydryl groups of ALA-D in a reduced state (essential for enzyme activity), since inhibition by these compounds was reversed by 10 mM dithiotreitol (a reducing agent). On the other hand, 10 mM dithiotreitol did not reverse ALA-D inhibition by a higher concentration of EDTA (100 microM). Accordingly, 2,3-dimercaptopropanol appears to inhibit ALA-D through a mechanism similar to that of low EDTA concentrations. Neither oxidized 2,3-dimercaptopropanol nor reactive oxygen species appeared to contribute for ALA-D inhibition by reduced 2,3-dimercaptopropanol. Taken together, these results suggest that 2,3-dimercaptopropanol inhibits ALA-D by chelating Zn2+ from a labile site that is involved in maintaining enzyme sulfhydryl groups in a reduced state. This site is compatible with the ZnB or Zn beta previously described in mammalian and bacterial ALA-D.

A enzima delta-aminolevullnato desidratase

Delta-aminolevulinic acid dehydratase (ALA-D, EC 4.2.1.24) is a sulfhydryl-containing enzyme that asymetrically condenses two molecules of delta-aminolevulinic acid (ALA), catalyzing the formation of porphobilinogen, the monopyrrole precursor of ali biological tetrapyrroles (corrins, porphyrins, chlorins). The two ALA molecules have been termed Aside ALA and P-side ALA in reference to their fates as the acetyl and propionyl halves of the product. P-side ALA binds first and forms a Schiffbase with an active-site Iysine. ALA-D is a cytosolic enzyme present in mammals, plants, fungi and bacteria. Bovine enzyme has a molecular mass of 280 000 Da and is composed of eight similar subunits of 35 000 Da, but only four of the subunits form a Schiff-base with the substrate (half-site reactivity). ALA-D from all organisms requires a bivalent metal ion for activity. Although the considerable sequence conservation among ALA-D enzyme from various organisms, there are species-dependent differences in metal ion requirements for enzyme activity. ALA-D is a zinc-dependent enzyme in animals, yeast and some bacteria. Mammalian enzyme bounds 8 zinc ions/octamer. Bovine ALA-D contains two types of Zn2+ binding sites (A and B), each at a stoichiometry of four per octamer. A-metal-ion-binding sites, with a single cysteine residue among its ligands, bind the four zinc ions essential for ALA-D activity (catalytic zinc), which plays a role in A-side ALA binding, in inter-ALA bond formation and in product binding. B-metal-ion-binding sites, with four cysteine residues among its ligands, bind zinc ions refered to as structural, which seems to be involved in the protection of sulfhydryl groups from oxidation. An A-zinc-ion-binding site has been proposed to be present at a number of four per octamer on the enzyme from plants, but has not been demonstrated yet. ALA-D from plants contains two types of magnesium binding sites: four B-metal-ion-binding sites (bind magnesium essential for ALA-D activity) and eight C-metal-ion-binding sites (bind magnesium that activates the enzyme but is not essential for activity). The cysteine-rich sequence of mammalian ALA-D that presumably corresponds to the B-metalion-binding site is replaced by an aspartate-rich sequence in plant ALA-D, probably accounting for the difference on metal-ion requirement (Mg2+ instead of Zn2+ on B-metal-ion-binding site from plant ALA-D). E. coli ALA-D binds eight Zn2+ (presumably four at A-metal-ion-binding site and four at B-metalion-binding site) and eight Mg2+ (presumably at C-metal-ion-binding site) per octamer. Due to its sulfhydrilic nature ALA-D is inhibited by heavy metals such as lead and mercury, serving as a measure of metal intoxication. In addition the inhibition of this enzyme has been implicated with pathological changes observed in some types of porphyrias, hepatorenal tyrosinemia and after lead or mercury exposure. ALA-D inhibition may impair haeme biosynthesis and leads to ALA accumulation, which besides being a potent agonist of y-aminobutiric acid autoreceptors may act as a prooxidant.