Interaction of zinc and other metal on the activity of erythrocyte delta-aminolevulinic acid dehydratase in vitro.

The interaction of zinc and other metal such as lead, mercury (II), cadmium, silver (II) on the activity of erythrocyte delta-aminolevulinic acid dehydratase (ALA-D) was investigated in vitro. At the concentrations ranging from 0.01 to 1.0 mmol/l, lead, mercury (II), cadmium and silver (II) strikingly inhibited the erythrocyte ALA-D activity. The in vitro addition of zinc having an activating effect for the erythrocyte ALA-D, was observed to eliminate the lead-induced inhibition of the erythrocyte ALA-D activity. And the degree of the elimination seemed to depend on the molar ratio (Zn/Pb) of both metal concentrations in the ALA-D assay. However, the inhibition of erythrocyte ALA-D activity caused by mercury (II), cadmium and silver (II) was not removed by the in vitro addition of zinc.     

Comparison of inhibition of erythrocyte pyrimidine 5'-nucleotidase and delta-aminolevulinic acid dehydratase by lead

The effect of lead on the activities of erythrocyte pyrimidine 5'-nucleotidase (P5N) and delta-aminolevulinic acid dehydratase (ALAD) was investigated in control and lead-exposed workers. The inhibitory effect of lead in vivo and in vitro was more remarkable on the ALAD than on the P5N activity values. It was demonstrated that the pH optimum of both ALAD and P5N in lead workers shifts to the acidic side (nearly pH 6.0) compared to that in control workers.     

Relationship between lead concentration in blood and biological response for porphyrin metabolism in workers occupationally exposed to lead

Summary The biological responses of the heme biosynthesis pathway in male workers moderately exposed to lead are discussed in relation to the concentration of lead in the blood. The level of erythrocyte delta-aminolevulinic acid dehydratase (ALA-D) activity in the group of lead-exposed workers was remarkably reduced while the level of erythrocyte protoporphyrin (Proto) in them was strikingly increased, compared to normal levels. On the other hand, the amounts of hemoglobin (Hb) and urinary delta-aminolevulinic acid (ALA) in the group of lead-exposed workers kept the normal levels.In the workers moderately exposed to lead, the log of erythrocyte Proto level was closely correlated to the blood lead level and the sensitivity of the Proto test was almost equal to that of erythrocyte ALA-D test. It was observed that the erythrocyte Proto was remarkably increased even in lead-exposed workers whose ALA excretion into the urine was in the range of normal level.     

Associations of lead exposure and dose measures with erythrocyte protein kinase C activity in 212 current Korean lead workers.

Lead can replace calcium in enzyme assays that measure protein kinase C activity and lead activates protein kinase C in human erythrocytes after exposure to lead in vitro. To examine the relevance of these observations to lead exposure in humans, we studied the associations of lead found in blood or tibia with activation of protein kinase C in erythrocytes isolated from workers in the lead industry. We examined erythrocytes among 212 lead workers, with a mean (+/-SD) age of 39.1 (10.0) years and exposure duration of 8.1 (6.5) years and measured protein kinase C activation by an in vitro back-phosphorylation assay. After adjustment for potential confounding factors (age and sex), tibia lead and exposure duration were significantly associated with erythrocyte protein kinase C activation (both p values < 0.05). No associations were observed between protein kinase C activation and blood-lead or zinc-protoporphyrin levels. These findings suggest that human exposure to lead results in activation of erythrocyte protein kinase C, which may be directly relevant to the neurotoxicity of lead.     

Relationship between activation of delta-aminolevulinic acid dehydratase by heating and blood lead level

Both blood lead and erythrocyte delta-aminolevulinic acid dehydratase (ALA-D) activity were determined for workers with and without an occupational lead exposure.In workers occupationally exposed to lead, it was demonstrated that the erythrocyte ALA-D is markedly activated by heating the hemolysate at 60° C for 5 min and there is a good positive correlation between the ratio of heated to nonheated ALA-D activity and the blood lead level (r = 0.799). In addition, by heating the hemolysate, the ALA-D activity of the lead-exposed workers appears to be returned into the normal range regardless of the extent of lead absorption. However, in normal workers without the occupational lead exposure, no significant correlation was found between the ratio of heated to nonheated ALA-D activity and the blood lead level, although the normal ALA-D also can be slightly activated by heating the hemolysate at 60° C for 5 min.     

Erythrocyte aminolevulinic acid dehydratase inhibition by cis-platin

The effect of cis-platin on erythrocyte aminolevulinic acid dehydratase (ALAD) activity was studied in vivo and in vitro. Young male Wistar rats were treated with a single i.p. injection of cis-platin at 2.5, 5.0, and 10.0mg/kg dose. In addition, a single i.p. injection of lead nitrate (1.0mg/kg dose) was administered as positive control. Experiments in vitro were also performed to elucidate the possible mechanism of action. The aminolevulinic acid dehydratase was almost completely inhibited in vitro from 0.5mM concentration, and the IC(50) was stated at 0.265 mM, 20 times higher than lead (IC(50) stated at 0.013 mM). Reduced glutathione, partially but significantly, reactivated in vitro the enzyme treated with cis-platin (0.5 and 5.0mM), whereas zinc showed a positive, significant effect with the higher dose (5.0mM) only. On the contrary, inhibition caused by lead (0.005 mM) was partially, but significantly restored by reduced glutathione, and, almost completely, by zinc. The experiments in vivo show that cis-platin causes a dose- and time-dependent inhibition of ALAD activity with 5.0 and 10.0mg/kg dose, until 66 and 33% of the control activity 96 h after treatment, respectively. The results show that erythrocyte ALAD is sensitive to cis-platin and suggest that the mechanism of enzyme inhibition is a direct interaction with sulfhydryl groups, whereas zinc site appears involved with the higher doses only. This mechanism appears different from lead that prevalently inhibits ALAD removing zinc from the enzyme, other than interacting with sulfhydryl groups.     

Comparative study of effects of lead on the activity of erythrocyte pyrimidine 5′-nucleotidase and delta-aminolevulinate dehydratase in vivo and in vitro

The effect of lead on the activities of erythrocyte pyrimidine 5′-nucleotidase (Py5N) and delta-aminolevulinate dehydratase (ALA-D) was studied in the mice which were given ad libitum a drinking water containing lead of 10, 50 and 250 ppm, for 27 days. The erythrocyte Py5N activity was not decreased in all groups of lead-exposed mice. However, the erythrocyte ALA-D activity was markedly decreased in the groups exposed to 50 and 250 ppm lead. These data indicate that erythrocyte ALA-D is more sensitive than Py5N to lead in vivo. On the other hand, from the in vitro study, it was demonstrated that the human erythrocyte Py5N is moderately inhibited by zinc and tin, and markedly by mercury, cadmium, silver, copper, and lead, at 10⁻⁴ molar concentrations. In addition, it was observed that the erythrocyte Py5N is most remarkably inhibited by mercury while the ALA-D by lead, among metals tested.     

Effect of tin on porphyrin biosynthesis

Certain disturbances in porphyrin biosynthesis were examined in rabbits administered with either tin or lead. Stannous chloride treatment increased the concentrations of coproporphyrin in blood and urine, as did lead acetate treatment. No effect of tin on 5-aminolevulinic acid concentration was observed in blood and urine, whereas lead treatment increased it markedly in both. Tin profoundly inhibited the activity of 5-aminolevulinate dehydratase in blood, but did not alter it in liver although tin content in liver was considerably higher than that in blood. In contrast to a prolonged inhibition of erythrocyte 5-aminolevulinate dehydratase by lead, tin inhibition of erythrocyte 5-aminolevulinate dehydratase activity was rapidly reversed after the cessation of the metal treatment.     

Combined effects of lead and EDTA on Na+,K+-ATPase activity of erythrocyte membranes

Erythrocyte Na+,K+-ATPase activity increased significantly in lead workers of a lead refining factory when measured with EDTA and compared to the controls without EDTA. The enzyme activity measured with EDTA increased in the following order: controls less than office workers in a lead refining factory less than lead workers. A positive correlation existed between blood lead and enzyme activity with EDTA (r = 0.380, p less than 0.10), and the activity without EDTA (r = 0.398, p less than 0.05). A negative correlation was found between sodium in erythrocytes and enzyme activity with EDTA (r = -0.437, p less than 0.05), and the activity without EDTA (r = -0.416). But no relationship was observed between enzyme activities and potassium in erythrocytes. A positive correlation between enzyme activity with EDTA and that without EDTA was observed (r = 0.452, p less than 0.05). With addition of lead to fragments of erythrocyte membranes, a significant decrease occurred in the activity of the enzyme without EDTA, whereas no change was observed with EDTA. No significant change occurred in the enzyme activity with and without EDTA upon addition of lead to blood. The maximum level of lead in membrane fragments (lead combined with membranes) of workers exposed to lead was 0.60 microgram/mg protein, and that in the experiment of addition to blood was 7.0 micrograms/mg protein.     

Erythrocyte acetylcholinesterase activity as a surrogate indicator of lead-induced neurotoxicity in occupational lead exposure in Abeokuta, Nigeria

Dose-effect and dose-response relationships in occupational neurotoxicology are rarely studied by means of biochemical methods. In order to investigate the potential neurotoxic effects of lead during occupational exposure to this metal, the activity of erythrocyte acetylcholinesterase (AcChE), as well as blood pressure and pulse, were determined in various artisans in Abeokuta, Nigeria, who have been shown to be occupationally exposed to lead, and these were related to blood lead levels. AcChE activity in the artisans was inhibited to varying extents. While AcChE activity was inhibited to the tune of 39% in the male petrol station attendants, the inhibition amounted to 32% in female petrol station attendants. In other artisans, AcChE inhibition ranged from 31% in the welders to 38% in painters. The lowest inhibition of 15% was obtained in the panel beaters. Correlations, as calculated by Pearson's method, revealed a significant (p<0.001) inverse linear relationship between AcChE activity and blood lead levels (r=-0.40; y=-120.38x+13935.59; p<0.001). Blood pressure and pulse were not significantly different between control and lead-exposed subjects. Our findings suggest that erythrocyte AcChE activity could be used as a biomarker of lead-induced neurotoxicity in occupationally exposed subjects.     

The action of small doses of lead on erythrocyte D-aminolevulinic acid dehydratase in the mouse

After a single intraperitoneal administration of lead in very small doses [1–100 g Pbac/kg body weight (bw)], there was a dose-dependent, highly significant inhibition of erythrocyte D-aminolevulinic acid dehydratase (D-ALA-D) activity in mice. The maximal inhibition occurred between 3 and 24 h post injection (p.i.). After that, a rapid recovery of the D-ALA-D activity took place so that four days after lead administration, enzyme activity exceeded even the normal value. Only after eight days p.i. did the D-ALA-D value return to the initial level after a biphasic course.After 10 i.p. injections of 0.1 to 10 g Pbac/kg bw, there was again a dose-dependent, highly significant inhibition of the erythrocyte D-ALA-D activity in mice. The maximal inhibition was shown to be 24 h after the last lead injection. In contrast to the single i.p. administration, however, we found a monophasic course for the return of D-ALA-D activity. The D-ALA-D values did not exceed the normal range at any time after 10 i.p. lead injections. Ten and 30 days oral administration of lead corresponding to i.p. doses exhibited similar results in D-ALA-D inhibition.     

Relative inhibition of rat plasma and erythrocyte cholinesterases by pesticide combinations

A selected combination of carbamate pesticides (carbofuran, oxamyl and propoxur were examined for cholinesterase (ChE) inhibition. The enzyme source was rat plasma and erythrocytes. Enzyme activity was determined colorimetrically using the Ellman technique adapted for erythrocyte ChE activity. Enzyme inhibition was obtained for both plasma and erythrocyte ChE, but inhibition by the pesticides was greater in the plasma. All 3 pesticides interacted additively in vitro.     

Correlation between clinical indicators of lead poisoning and oxidative stress parameters in controls and lead-exposed workers

The present study was undertaken to investigate the involvement of oxidative damage in lead-induced toxicity in humans and to enlighten whether oxidative stress indicators are correlated with the known indices of lead toxicity. For these purposes, selected oxidative stress parameters along with some clinical indices of lead poisoning were determined in blood of battery plant workers and control subjects. Workers had significantly increased erythrocyte malondialdehyde (MDA) levels, catalase and glucose-6-phosphate dehydrogenase (G6PD) activities, and decreased blood glutathione:glutathione disulfide ratio compared to the controls. Increased blood lead concentrations and zinc protoporphyrin (ZPP) levels, and decreased delta-aminolevulinic acid dehydratase (ALAD) activity were used as clinical indices of lead toxicity. Statistically significant correlation between oxidative stress parameters and clinical indices implies that disrupted prooxidant/antioxidant balance might contribute to lead-induced toxicity in erythrocytes. A significant correlation was found between ALAD activity and blood lead levels in human subjects. Similarly significant correlation between ALAD activity and erythrocyte MDA concentrations was shown. Present data indicates that ALAD can serve as a valuable biomarker of oxidative stress in lead-exposed hematological system as well as being a biochemical indicator of lead exposure.     

Lysophosphatidylcholine hydrolases of human erythrocytes, lymphocytes, and brain: sensitive targets of conserved specificity for organophosphorus delayed neurotoxicants

Brain neuropathy target esterase (NTE), associated with organophosphorus (OP)-induced delayed neuropathy, has the same OP inhibitor sensitivity and specificity profiles assayed in the classical way (paraoxon-resistant, mipafox-sensitive hydrolysis of phenyl valerate) or with lysophosphatidylcholine (LysoPC) as the substrate. Extending our earlier observation with mice, we now examine human erythrocyte, lymphocyte, and brain LysoPC hydrolases as possible sensitive targets for OP delayed neurotoxicants and insecticides. Inhibitor profiling of human erythrocytes and lymphocytes gave the surprising result of essentially the same pattern as with brain. Human erythrocyte LysoPC hydrolases are highly sensitive to OP delayed neurotoxicants, with in vitro IC50 values of 0.13-85 nM for longer alkyl analogs, and poorly sensitive to the current OP insecticides. In agricultural workers, erythrocyte LysoPC hydrolyzing activities are similar for newborn children and their mothers and do not vary with paraoxonase status but have high intersample variation that limits their use as a biomarker. Mouse erythrocyte LysoPC hydrolase activity is also of low sensitivity in vitro and in vivo to the OP insecticides whereas the delayed neurotoxicant ethyl n-octylphosphonyl fluoride inhibits activity in vivo at 1-3 mg/kg. Overall, inhibition of blood LysoPC hydrolases is as good as inhibition of brain NTE as a predictor of OP inducers of delayed neuropathy. NTE and lysophospholipases (LysoPLAs) both hydrolyze LysoPC, yet they are in distinct enzyme families with no sequence homology and very different catalytic sites. The relative contributions of NTE and LysoPLAs to LysoPC hydrolysis and clearance from erythrocytes, lymphocytes, and brain remain to be defined.     

Lignocaine: inhibitory effect on synaptosomal and erythrocyte membrane-bound acetylcholinesterase activity

Lignocaine (5-20 mM) reversibly inhibits (in vitro) AChE activity of rat brain synaptosome (33-66%) and erythrocyte membrane (10-54%) in a concentration dependent manner. Lineweaver-Burk plots indicate that lignocaine-induced inhibition of AChE activity in synaptosome is competitive, whereas in erythrocyte membrane inhibition of AChE is non-competitive in nature. Arrhenius plots show that transition temperatures of both synaptosomal and erythrocyte membrane-bound AChE are significantly reduced in the presence of lignocaine. These results suggest that lignocaine increases the lipid fluidity of synaptosomal and erythrocyte membrane which may be a cause of inhibition of membrane-bound AChE activity.     

In vitro kinetic interactions of pyridostigmine, physostigmine and soman with erythrocyte and muscle acetylcholinesterase from different species

The low effectiveness of atropine and oxime treatment in soman poisoning may be enhanced by carbamates pre-treatment. For ethical reasons medical countermeasures can only be tested in animal models despite the fact of substantial species differences. With this kinetic in vitro study the interactions between pyridostigmine, physostigmine and soman with human, Rhesus monkey, swine and guinea pig erythrocyte AChE were investigated. In addition, the effect of the carbamates on the residual activity and enzyme recovery after soman inhibition was examined with erythrocyte and intercostal muscle AChE from these species with a dynamic in vitro model with real-time determination of AChE activity. Only small to moderate species differences of the inhibition and decarbamylation kinetics were recorded. It was possible to show that with erythrocyte and muscle AChE a similar level of protection by carbamates and reactivation after discontinuation of the carbamates and soman could be observed. Thus, these data indicate that carbamate pre-treatment is expected to protect a critical level of muscle AChE and confirm the presumption that erythrocyte AChE may serve as a surrogate for synaptic AChE. Hence, these and previous data fortify the notion that erythrocyte AChE is a proper tool for in vitro kinetic studies as well as for therapeutic monitoring in experimental and clinical studies.     

Erythrocyte enzymes in experimental lead poisoning

Intravenous administration of 6 mg per kg lead acetate to rabbits resulted in plumbism with elevated erythrocyte lead levels and marked depression of activity of erythrocyte -aminolevulmic acid dehydratase. By comparison other erythrocyte enzymes were insensitive to the effects of lead. Activities of anaerobic glycolysis and of the hexose monophosphate shunt were unaffected by lead administration as were erythrocyte methemoglobin reductase, acid phosphatase, glucose-6-phosphate dehydrogenase, malic dehydrogenase and acetylcholinesterase. The insensitivity of these erythrocyte enzymes to inhibition by lead excludes their usefulness for detection or diagnosis of plumbism.     

Comparative kinetics of organophosphates and oximes with erythrocyte, muscle and brain acetylcholinesterase

There is an ongoing debate whether oximes can effectively counteract the effects of organophosphorus compounds (OP) on brain acetylcholinesterase (AChE) activity and whether there are differences in the kinetic properties of brain and erythrocyte AChE. In order to investigate the kinetics of AChE from different tissues and species the well established dynamically working in vitro model with real-time determination of membrane-bound AChE activity was adapted for use with brain AChE. The enzyme reactor, that was loaded with brain, erythrocyte or muscle AChE, was continuously perfused with substrate and chromogen while AChE activity was on-line analyzed in a flow-through detector. It was possible to determine the Michaelis-Menten constants of human erythrocyte, muscle and brain AChE which were almost identical. In addition, the inhibition kinetics of sarin and paraoxon as well as the reactivation kinetics of obidoxime and HI 6 were determined with human, swine and guinea pig brain and erythrocyte AChE. It was found that the inhibition and reactivation kinetics of brain and erythrocyte AChE were highly comparable in all tested species. These data support the view that AChE from different tissue has similar kinetic properties and that brain AChE is comparably susceptible toward reactivation by oximes.     

Decreased erythrocyte delta-aminolevulinate dehydratase activity after styrene exposure

delta-Aminolevulinate dehydratase (ALA-D:porphobilinogen synthase, 5-aminolevulinate hydro-lyase, EC 4.2.1.24) activity was depressed markedly in red cells of rats exposed to 0.21 g/m3 styrene, a chemical widely used in commercial products. The depression was not restored in vitro after treatment with dithiothreitol and zinc. Consistent with this finding, radioimmunoassay of the enzyme protein demonstrated reduction in the enzyme concentration by styrene exposure. There was a good correlation between the decrease in enzyme activity and its concentration in the styrene-treated animals, suggesting that the depression of the enzyme activity was essentially due to the reduction in the enzyme content. Decrease in the enzyme content in bone marrow cells to almost the same extent as that in erythrocytes seems to indicate the decreased synthesis of ALA-D in the bone marrow. In vitro studies showed that styrene 7,8-oxide, the major intermediate of styrene metabolism, decreased the activity of purified ALA-D but that styrene, the parent compound itself, had no inhibitory effect. The activity and concentration of erythrocyte ALA-D in workers chronically exposed to styrene were also depressed significantly. These findings indicate that the styrene exposure-mediated decrease of ALA-D activity in erythrocytes was a reflection of reduction in the enzyme protein, which may have been the result of styrene 7,8-oxide action, and they suggest that a similar process may also be involved in the reduction of erythrocyte ALA-D in styrene-exposed workers.     

Delta-aminolaevulinic acid metabolism in normal and lead-exposed humans

The activity of the haem biosynthetic enzymes delta-aminolaevulinic acid synthetase (ALA.S) and delta-aminolaevulinic acid dehydratase (ALA.D) were measured in the peripheral blood of a group of lead workers and control subjects. The haem precursor delta-aminolaevulinic acid (ALA) was measured in blood and urine, whilst lead levels were measured in whole blood. The inter-relationships between all these parameters were examined and quantified. The results demonstrate that above a blood lead concentration of 2 mumole/l and below an erythrocyte ALA.D activity of 18 nmole ALA utlized/min/ml red blood cells (R.B.C.), Haem synthesis is depressed to such an extent that the activity of leucocyte ALA.S, the rate-limiting enzyme of haem biosynthesis, is increased by negative feedback.