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High levels of methionine (Met) and methionine sulfoxide (MetO) are found in several genetic abnormalities. Oxidative stress is involved in the pathophysiology of many inborn errors of metabolism. However, little is known about the role of oxidative damage in hepatic and renal changes in hypermethioninemia. We investigated the effect of chronic treatment with Met and/or MetO on oxidative stress parameters in liver and kidney, as lipid peroxidation (TBARS), total sulfhydryl content (SH), reactive oxygen species (ROS) and enzymes activities superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and delta aminolevulinic dehydratase (ALA-D). Serum biochemical parameters were evaluated. Wistar rats were treated daily with two subcutaneous injections of saline (control), Met (0.2-0.4 g/kg), MetO (0.05-0.1 g/kg) and the association between these (Met plus MetO) from the 6th to the 28th day of life. Our data demonstrated an increase of glucose and urea levels in all experimental groups. Cholesterol (MetO and Met plus MetO) were decreased and triglycerides (MetO) were increased. SOD (MetO and Met plus MetO) and CAT (Met, MetO and Met plus MetO) activities were decreased, while GPx was enhanced by MetO and Met plus MetO treatment in liver. In kidney, we observed a reduction of SH levels, SOD and CAT activities and an increase of TBARS levels in all experimental groups. ROS levels in kidney were increased in MetO and Met plus MetO groups. ALA-D activity was enhanced in liver (MetO and Met plus MetO) and kidney (Met plus MetO). These findings help to understand the pathophysiology of hepatic and renal alterations present in hypermethioninemia.

Lead (Pb(2+)) is a heavy metal that has long been used by humans for a wide range of technological purposes, which is the main reason for its current widespread distribution. Pb(2+) is thought to enter erythrocytes through anion exchange and to remain in the cell by binding to thiol groups. Pyruvate kinase (PK) is a thiol-containing enzyme that plays a key role in erythrocyte cellular energy homeostasis. δ-aminolevulinic acid dehydratase (δ-ALAD) is the second enzyme in the heme biosynthetic pathway and plays a role in the pathogenesis of Pb poisoning. Our primary objective was to investigate the effect of Pb(2+) on the activity of the thiolenzymes δ-ALAD and PK and on the concentration of glutathione (GSH), a nonenzymatic antioxidant defense, in erythrocytes from Pb-exposed workers. The study sample comprised 22 male Pb workers and 21 normal volunteers (15 men and 6 women). The Pb-exposed workers were employed in manufacturing and recycling of automotive batteries. Basic red-cell parameters were assayed and total white blood cell counts performed. PK and δ-ALAD activity and blood Pb (BPb) concentrations were determined in all subjects. Pb-exposed individuals had significantly greater BPb levels than controls. Both PK and δ-ALAD activity levels were significantly lower in Pb-exposed individuals than in controls. Pb significantly inhibited PK and δ-ALAD activity in a dose-dependent manner. We found that erythrocyte GSH levels were lower in Pb-exposed individuals than normal volunteers. Pb-exposed individuals had lower values than controls for several red cell parameters (hemoglobin, hematocrit, red blood cell count, mean corpuscular volume). These results suggest that Pb inhibits δ-ALAD and PK activity by interacting with their thiol groups. It is therefore possible that Pb disrupts energy homeostasis and may be linked with decreased glucose metabolism because it affects the heme synthesis pathway in erythrocytes, contributing to the cell dysfunction observed in these in Pb-exposed individuals. These results indicate an apparent dose-effect relationship between PK activity and BPb. PK activity in human erythrocytes can be used for biological monitoring of Pb exposure. Study of the mechanisms by which Pb acts may contribute to greater understanding of the symptoms caused by Pb.

Child low-level lead (Pb) exposure is an unresolved public health problem and an unaddressed child health disparity. Particularly in cases of low-level exposure, source removal can be impossible to accomplish, and the only practical strategy for reducing risk may be primary prevention. Genetic biomarkers of increased neurotoxic risk could help to identify small subgroups of children for early intervention. Previous studies have suggested that, by way of a distinct mechanism, δ-aminolevulinic acid dehydratase single nucleotide polymorphism 2 (ALAD(2)) and/or peptide transporter 2*2 haplotype (hPEPT2*2) increase Pb blood burden in children. Studies have not yet examined whether sex mediates the effects of genotype on blood Pb burden. Also, previous studies have not included blood iron (Fe) level in their analyses. Blood and cheek cell samples were obtained from 306 minority children, ages 5.1 to 12.9 years. (208)Pb and (56)Fe levels were determined with inductively coupled plasma-mass spectrometry. General linear model analyses were used to examine differences in Pb blood burden by genotype and sex while controlling for blood Fe level. The sample geometric mean Pb level was 2.75 μg/dl. Pb blood burden was differentially higher in ALAD(2) heterozygous boys and hPEPT2*2 homozygous boys. These results suggest that the effect of ALAD(2) and hPEPT2*2 on Pb blood burden may be sexually dimorphic. ALAD(2) and hPEPT2*2 may be novel biomarkers of health and mental health risks in male children exposed to low levels of Pb., (© Springer Science+Business Media, LLC 2011)

The oligomeric state of human porphobilinogen synthase (PBGS) [EC.4.2.1.24] is homooctamer, which consists of conformationally heterogenous subunits in the tertiary structure under air-saturated conditions. When PBGS is activated by reducing agent with zinc ion, a reservoir zinc ion coordinated by Cys(223) is transferred in the active center to be coordinated by Cys(122), Cys(124), and Cys(132) (Sawada et al. in J Biol Inorg Chem 10:199-207, 2005). The latter zinc ion serves as an electrophilic catalysis. In this study, we investigated a conformational change associated with the PBGS activation by reducing agent and zinc ion using analytical ultracentrifugation, negative staining electron microscopy, native PAGE, and enzyme activity staining. The results are in good agreement with our notion that the main component of PBGS is octamer with a few percent of hexamer and that the octamer changes spatial subunit arrangement upon reduction and further addition of zinc ion, accompanying decrease in f/f (0). It is concluded that redox-regulated PBGS activation via cleavage of disulfide bonds among Cys(122), Cys(124), and Cys(132) and coordination with zinc ion is closely linked to change in the oligomeric state.

Oximes are a class of compounds normally used to reverse the acetylcholinesterase (AChE) inhibition caused by organophosphates (OPs). Conversely, researches focusing on the possible antioxidant properties of these compounds are lacking in the literature. The aim of this study was to investigate the potential antioxidant and toxic properties of 3-(phenylhydrazono) butan-2-one oxime in mice. In vitro, hydrogen peroxide-induced lipid peroxidation was decreased by low concentrations of the oxime (0.1-1.0 microM); (P < 0.05). Similarly, lipoperoxidation induced by malonate and iron (Fe2+) was significantly decreased by the oxime (0.4-1.0 microM) (P < 0.05). Oxime pre-treatment did not modify the basal peroxidation level nor prevented the induced lipid peroxidation determined ex-vivo. The present results suggest that 3-(phenylhydrazono) butan-2-one oxime could be a good antioxidant compound. The absence of toxicity signs after in vivo administration of 3-(phenylhydrazono) butan-2-one oxime to mice may indicate that it could be a safe drug for further studies.

Lead poisoning of waterfowl has been reported for decades in the Coeur d'Alene River Basin (CDARB) in Idaho as a result of the ingestion of lead-contaminated sediments. This study was conducted to determine whether the addition of phosphoric acid to sediments would reduce the bioavailability and toxicity of lead to mallards (Anas platyrhynchos) as related to adverse hematological effects and altered plasma chemistries. Mallards received diets containing 12% clean sediment (controls) or 12% sediment from three different CDARB sites containing 4520, 5390, or 6990 microg/g lead (dw) with or without phosphoric acid amendment. Blood lead concentrations were significantly higher in all CDARB treatment groups and ranged from geometric mean values of 5.0 microg/g for the first two sites to 6.2 microg/g for the third site. With amendments, all blood lead concentrations became 41% to 64% lower. Red blood cell ALAD activity was depressed by 90% or more with lead-contaminated sediment from all sites and did not differ with amended diets. Free erythrocyte protoporphyrin (FEP) concentrations were elevated by contaminated sediment from all sites. Amendment decreased the elevations in FEP by as much as 80%. Hematocrit values and hemoglobin concentrations were lower for all lead site sediments by as much as 30% for site 3. Plasma enzyme activities for ALT, CK, and LDH-L were elevated by as much as 2.2-fold, and plasma creatinine concentration was 1.7-fold higher for site 3 sediment. Amendments restored hematocrit, hemoglobin, and plasma enzyme activities so that they did not differ from controls. Although amendments of phosphorus substantially reduced the bioavailability of lead and alleviated many of the adverse hematological effects, lead concentrations in the blood of mallards fed the amended sediments were still above those believed to be harmful to waterfowl under the present conditions.

An initial survey of lead levels in American woodcock (Scolopax minor) from Wisconsin was conducted in 1998 using wing bones from hunter-donated woodcock. The results of this initial survey indicated that young-of-year woodcock were accumulating extremely high levels of lead in their bones. Similar collections were made (using steel shot) between 1999 and 2001. The combined results of this collection indicated that 43.4% of young-of-year woodcock (range 1.5-220.0 microg/g dry wt) and 70% of woodcock chicks (range 9.6-93.0 microg/g dry wt) had bone lead levels in the elevated range (>20 microg/g dry wt). Blood samples were collected from chicks at a site considered elevated based on bone lead results (Mead Wildlife Area) and a site considered background (Navarino Wildlife Area). These samples were analyzed for lead concentration and aminolevulinic acid dehydratase activity. The mean blood lead concentrations of woodcock chicks from both sites did not reach levels that are considered elevated in waterfowl (>0.200 microg/ml). However, blood lead concentrations of chicks from the Mead Wildlife Area were significantly higher than lead levels in chicks from Navarino Wildlife Area (p = 0.002). Although the ultimate sources of lead exposure for Wisconsin woodcock currently remain unidentified, anthropogenic sources cannot be ruled out. Our results indicate that elevated lead exposure in Wisconsin woodcock is common and begins shortly after hatch.

Lead (Pb) is a ubiquitous environmental pollutant, widely distributed, representing a high toxicological and ecotoxicological risk. Several morphological, functional, and biochemical parameters have been proposed as biomarkers of effect and exposure to Pb. The information related to adverse effects of Pb is not abundant for adult amphibians. These animals are of interest, because during their development they move from aquatic to terrestrial habitats, which may be polluted by the metal since they are receptors of products generated by anthropogenic activities. Previous studies carried out on the adult South American toad Bufo arenarum (Amphibia, Anura) showed that it has a high tolerance to lead and studied the effect of sublethal doses of the metal on the erythrocyte osmotic fragility and delta-ALAD activity. It was also shown that after a single injection of Pb, a significant increase in the number of reticulocytes was produced, suggesting the suitability of those cell counts as a biomarker of exposure to the metal; its impact on the immune system of the toads was also studied. In this work we extend our early studies on the same species evaluating the chronic effect of sublethal Pb (equivalent to 5.6% of the 120-h LD-50) on free erythrocyte protoporphyrin (FEP) and blood Pb and delta-ALAD activity; blood lead was positively associated with a significant decrease in the enzyme activity and to an increase in the FEP level. Pb concentration in target organs (liver, spleen, femur, and kidney) and the total cumulated amount as well as its impact over the mass of those organs were also determined. In addition, the magnitude of the possible depuration through urine and intestine was evaluated. Our results showed that FEP, delta-ALAD, and blood Pb are reliable biosensors of chronic metal intoxication, the former being the marker with the highest sensitivity.

Inorganic lead exposure was studied in 31 volunteers employed in storage battery plant. The genotoxicity of lead was measured in terms of sister chromatid exchange (SCE). Erythrocyte delta-aminolevulinic acid dehydrogenase (ALAD) activity, urinary delta-aminolevulinic acid (U-ALA), and blood lead levels (PbBs) were also determined to evaluate some possible relations between these lead exposure indicators and the observed SCE frequencies. Blood lead concentration of 36.31 microg/dl was determined as an average level in the workers. Consequently decreased ALAD activity in erythrocytes and increased U-ALA excretion was observed in statistically higher PbBs when compared with the control group. A statistically significant correlation was observed between the PbBs and SCE frequencies (p < 0.05). Moreover, the correlation between U-ALA excretion and SCE frequencies (p < 0.01) was relatively higher than the correlation between PbBs and SCE frequencies. These results might indicate a possible mechanism of ALA mediation in the genotoxic effects of lead.

Objective: To determine the effect of different durations of exposure to agricultural chemicals on the activities of the blood enzymes delta-aminolevulinic acid dehydratase (ALAD), superoxide dismutase (SOD), and cholinesterase (ChE) in tobacco field workers., Methods: For this preliminary investigation, 8 volunteers (all smoked tobacco) who were working on a small tobacco farm were monitored over a period of 2 years along with a comparable urban unexposed group (n = 4). During the growing season between 1994 and 1996, dermal and respiratory exposure were determined and blood samples were drawn after the following durations of field work: (1) preexposure (0 DAY); (2) after 1 day of field work (1 DAY) - workers reentered fields at 24 h after spraying of acephate and maleic hydrazide; (3) after 30 days of field work (postspraying; 30 DAYS); and (4) Postexposure - no tobacco production. Standard analytical methods were used., Results: Activity of ALAD was depressed by 30% after 1 DAY and there was no further decrease in ALAD activity after 30 DAYS of field work. SOD activity, in contrast, declined by 29% and 50% after 1 DAY and 30 DAYS, respectively, as compared with 0-DAY activity and that of the urban control, which was similar to 0-DAY activity (P< or =0.05). Plasma ChE activity declined by 19% after both 1 and 30 DAYS of exposure/field work. The activities of all three enzymes were restored to urban control or preexposure levels during postexposure. Plasma Cd levels were high in the samples taken after 30 DAYS as compared with the preexposure levels. Respiratory nicotine exposure was highest after 30 DAYS of field work., Conclusion: This preliminary study suggests that erythrocyte SOD is a sensitive indicator of exposure to agricultural chemicals in tobacco field workers.

During tetrapyrrole biosynthesis 5-aminolevulinic acid dehydratase (ALAD) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA) to form one molecule of the pyrrole derivative porphobilinogen. In Escherichia coli, the enzyme is encoded by the gene hemB. The hemB gene was cloned from Pseudomonas aeruginosa by functional complementation of an E. coli hemB mutant. An open reading frame of 1011 bp encoding a protein of 336 amino acids (M(r) 37,008) was identified. The gene was mapped to SpeI fragment G and DpnI fragment G of the P. aeruginosa chromosome, corresponding to the 10 to 12 min region of the new map or 19 to 22 min interval of the old map. The 5' end of the hemB mRNA was determined and the -10 and -35 regions of a potential sigma 70-dependent promoter were localized. No obvious regulation of the hemB gene by oxygen, nitrate, heme or iron was detected. Alignment of the amino acid sequences deduced from hemB revealed a potential metal-binding site and indicated that the enzyme is Mg(2+)-dependent. P. aeruginosa hemB was overexpressed in an E. coli hemB mutant using the phage T7 RNA polymerase system and its Mg(2+)-dependent activity was directly demonstrated.

This study was performed to evaluate the bioavailability and effects of lead in wildlife at a trap and skeet range. The total lead concentration in a composite soil sample (pellets removed) was 75,000 micrograms/g dry weight. Elevated tissue lead concentrations and depressed ALAD activities in small mammals and frogs indicate that some of the lead deposited at the site is bioavailable. Mean tissue lead concentrations (micrograms/g dry wt.) in white-footed mice (Peromyscus leucopus) at the range liver = 4.98, kidney = 34.9, femur = 245) were elevated (P < 0.01) 5- to 64-fold relative to concentrations in mice from a control area. Tissue lead concentrations in the only shorttail shrew (Blarina brevicauda) captured at the range (liver = 34.1, kidney = 1506, femur = 437) were elevated 35- to 1038-fold. Femur lead concentrations in green frogs (Rana clamitans) at the range (1,728 micrograms/g) were elevated nearly 1000-fold, and the lead concentration in a pooled kidney sample (96.2 micrograms/g) was elevated 67-fold. There was significant depression of blood ALAD activity in mice (P = 0.0384) and depression of blood and liver ALAD activity in frogs (P < 0.001). Hematological and histopathological lesions associated with lead toxicosis were observed in some animals. Hemoglobin concentrations were reduced 6.7% in mice (P = 0.0249), but hematocrit was not significantly affected in mice or frogs. Intranuclear inclusions were present in the renal proximal tubular epithelium of two of the mice and the shrew that were captured at the range, and necrosis of the tubular epithelium was also evident in one of the mice. Kidney:body weight ratios were similar in range and control mice. Soil ingestion may be a significant route of lead uptake in small mammals at the range. However, the tendency of lead to concentrate in the bones rather than in more digestible soft tissues may minimize food chain uptake of lead by predators, especially raptors that regurgitate undigestible material.

To evaluate the subclinical effect of lead exposure, we determined delta-aminolevulinic acid (ALA) levels in plasma (ALA-P), blood (ALA-B), and urine (ALA-U) and the activity of delta-aminolevulinic acid dehydratase (ALAD) in lead workers. Almost all of the ALA molecules in blood were present in plasma and not in blood cells, irrespective of the blood lead concentration (Pb-B). ALA-P or ALA-B levels increased slowly at Pb-B levels below 40 micrograms/dl (slow phase) and rapidly at levels above 40 micrograms/dl (rapid phase). In both phases, ALA-P and ALA-B were well correlated with Pb-B and ALAD activity. The threshold value (no-effect level) of Pb-B for elevation of the ALA-P or ALA-B level was coincident with that for ALAD inhibition; the value was around 5 micrograms/dl. In the rapid phase, ALA-P increased continuously up to 100 micrograms/dl of Pb-B, while ALAD activity reached a plateau. Receiver operative characteristic (ROC) plot analyses indicated that ALA-P and ALAD activity [ALAD(u)] had a similar diagnostic value at Pb-B levels between 10 and 40 micrograms/dl, although ALAD(%), the remaining ALAD activity as a percentage of the whole activity restored by zinc and dithiothreitol, had the most powerful diagnostic efficiency at these Pb-B levels. By contrast, ALA-U and zinc protoporphyrin were less effective for the diagnosis of lead exposure than ALAD and ALA-P. These findings indicate that ALA-P is the best discriminators of lead exposure form baseline to high levels of exposure.

The activity of the enzyme delta-aminolevulinic acid dehydratase (ALA-D) was strongly depressed in erythrocytes, spleen and renal tissue of rainbow trouts (Salmo gairdnerii) exposed to sublethal levels of inorganic lead (10, 75 and 300 microgram Pb2+/L) for 30 days. The fish exposed to the highest lead concentration also showed an anemic response and basophilic stippling of erythrocytes. The lead exposure did not cause any alteration of the white blood cell picture. The ALA-D inhibition, the anemia and basophilic stippling seemed to persist in the lead-exposed fish even after a recovery period of seven weeks in lead-free water.

The delta-aminolevulinate dehydratase (ALA.D) quantitative assay on a centrifugal fast analyser showed that subjects whose blood-lead level varies between 30 and 75 micrograms/100 ml (1.5 to 3.75 microM/l) react to blood intoxication by synthesizing de novo an amount of enzyme correlating to blood-lead levels. At higher concentrations, the reactional synthesis occurs very rarely. These results suggest that enzyme is constitutive, but also inductible as soon as its substrate accumulates; this last ability may disappear at high blood-lead levels: a hypothesis is proposed thereafter.

The effects of N,N'-methylene-bis-acrylamide (MBA), a cross-linking agent, on blood and bone marrow after repeated oral doses, were studied in mice and rats. Body weight, three major elements of the blood - erythrocytes, leucocytes and platelets -, reticulocytes and bone marrow cells, were all reduced in either or both animals, especially in mice. Phenobarbital (PB) treatment did not greatly modify the effects of MBA in mice. An increase in free erythrocyte porphyrins and a decrease in ALA-D activity were observed in both animals. Urinary porphyrins were elevated in rats after MBA-dosing. PB-treatment did not significantly affect the elevation of porphyrins. After cessation of the MBA-dosing, all these changes were inclined to be restored to normal levels. Amounts of liver total porphyrins and microsomal P-450, and red cell fragility were within normal ranges in mice.

The authors studied in vitro the variations of erythrocyte ALA D activity in subjects exposed and not exposed to lead risk before and after three tests: heat treatment at 60 degrees C for five minutes, addition of GSH (1 X 10(-2) mol/l) and of zinc (1.5 X 10(-4) mol/l). A study of the ratios before and after treatments showed that GSH and zinc were better than heating in identifying false positives, and that zinc was preferable, owing to lesser dispersion of results around the mean. The ratios of ALA D activity before and after treatment is more closely correlated with PbB levels than ALA D activity without treatment. The addition of GSH restored the enzyme activity of the exposed subjects to values equal to those obtained with heating; this did not happen in the non-exposed subjects. It is postulated that, in lead exposure, the metal acts only on the thiol groups, which may then be reactivated by an optimal concentration of GSH.

Despite the widespread use of hand-crafted lead-glazed pottery by the Arab population, no cases of lead intoxication have previously been attributed to these ceramics. Three Arab families were found that used to eat from such earthenware utensils and were afflicted with plumbism. Plumbism was diagnosed by the determination of blood and urine lead levels and in two of the families by the decrease of red cell delta-aminolevulinic acid dehydratase activity. The propositus of the first family was the 23-year-old mother who had abdominal pains and hematuria. Lead intoxication was established and therapy with calcium EDTA institutes. She recovered. Her one-year-old daughter died in another hospital during a similar episode. Family studies revealed subclinical plumbism in her remaining three sons. In another family, a 54-year-old-man had abdominal pains and had a lead line on the gingival margins. Subsequent testing of the surfaces of the earthenware containers from which these families used to eat disclosed excessive amounts of lead. In the third family, eight members had plumbism, four of them with encephalopathy associated with motor palsy. One case was icteric. They also used to eat from primitive pottery with a lead glaze.

In order to test neurobehavioral effects of low-level lead exposure during early development, Wistar-rats were given pre- and postnatal dietary lead as lead acetate in four concentrations: 0, 80, 250, and 750 ppm. These diets, known to give rise to blood lead-levels (PbB) of less than 5, 11, 18, and 31 micrograms/dl, resulted in erythrocyte ALAD-inhibition of 40, 73, and 83%, respectively. The animals were tested first at 70 to 100 days postnatal (PN 70-100) in a 2-way active avoidance-task, and then at PN 190-250 in a visual discrimination-task. Lead-exposure was associated with performance-disruption in the discrimination-task, significant (p less than 0.001) already for the 250 ppm-exposure, but with significant (p less than 0.05) performance-facilitation in the avoidance-task. If learning and retention in animals is taken as a measure of cognitive performance neurobehavioral lead-toxicity cannot easily be explained in terms of cognitive deficit. An interpretation in terms of emotional reactivity or behavioral disinhibition would seem more convincing, which covers the results from activity-studies as well. 70% ALAD-inhibition corresponds to PbBs of about 20 micrograms/dl in rats, and to about 40 micrograms/dl in children. Some implications of animal studies for neuropsychological results from Pb-exposed children are discussed.

To evaluate the effects of calcium disodium ethylenediamine tetraacetate (CaEDTA) on the concentrations of lead, zinc and copper in plasma, erythrocyte and urine, and the delta-aminolevulinic acid dehydratase (ALAD) activity in erythrocyte, we administered CaEDTA in 1-h intravenous infusion to ten male gun metal founders with blood-lead concentration of 39 to 64 micrograms/dl (mean 49 micrograms/dl). We found that the plasma concentration of lead, following a rapid rise within the first 3 h, fell temporarily to the level significantly lower than the initial level 19 h after start of the infusion. The plasma concentration of zinc fell to the minimal level 5 h after the infusion; and the erythrocyte concentration of zinc and the ALAD activity concurrently rose to the maximal level 5 h after the infusion. By contrast, no significant alteration was observed in the concentrations of copper in plasma and erythrocyte. The maximal level of urinary metal excretion was attained during the period between 1 and 2 h after start of CaEDTA infusion for lead; within 2 h for zinc; and between 2 and 4 h for copper. The urinary metal excretion returned to the initial level 14 to 24 h after infusion for zinc and copper; but lead excretion was still higher than the initial level during this period. The difference in the kinetics of the three metals following CaEDTA injection is discussed in the light of these findings.