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 treatment with mercury chloride and lead acetate during the second stage of rapid postnatal brain growth on δ-aminolevulinic acid dehydratase (ALA-D) activity in brain, liver, kidney and blood of suckling rats

The sensitivity of developing rodents to toxic metals differs considerably from that of adults. In the present study, we investigated the in vivo and in vitro effects of inorganic mercury and lead on δ-aminolevulinic acid dehydratase (ALA-D) from brain, liver, kidney and blood of young rats. Eight day-old rats were injected with one or five doses of lead acetate (0, 3.5, or 7.0 mg/kg) or HgCl₂ (0, 2.5, or 5.0 mg/kg). In vitro, the IC₅₀ for mercury inhibition of cerebral, renal and hepatic ALA-D was in the 124 to 160 μM range, while values for lead acetate was in the 7 to 12 μM range. The IC₅₀ of blood enzyme for lead (0.8 μM) and mercury (6.5 μM) was significantly lower than that observed for the other tissues. A single dose of lead did not affect the enzyme activity, but a single dose of HgCl₂ (5 mg/kg) caused a significant inhibition of ALA-D from kidney (40%, P < 0.01) and liver (25%, P < 0.05). Five doses of lead acetate (3.5 or 7 mg/kg) caused an inhibition of about 25 and 40%, respectively (P < 0.01), of hepatic ALA-D, and an increase of 1.4-fold (P < 0.05) and 2.6-fold (P < 0.01) of blood enzyme, respectively. Treatment with five doses of HgCl₂ (5 mg/kg) caused an inhibition of about 25, 60, 50, and 80% of ALA-D from brain, blood, liver and kidney, respectively (all P < 0.05). Five doses of 2.5 mg/kg HgCl₂ caused an inhibition of ALA-D from liver (40%, P < 0.01) and kidney (45%, P < 0.01). These results demonstrate that ALA-D from young rat tissues show different sensitivities to mercury and lead. The enzyme was more affected by mercury than by lead in vivo, while in vitro lead was more potent that mercury as an ALA-D inhibitor.     

A comparison of the stimulatory effects of cadmium and zinc on normal and lead-inhibited human erythrocytic delta-aminolevulinic acid dehydratase activity in vitro

Zinc has been reported to activate δ-aminolevulinic acid dehydratase (ALAD) and to reverse lead-induced inhibition of human blood ALAD activity in vitro. Inasmuch as cadmium lies just below zinc in Group IIB of the periodic table of elements, similar studies were initiated with cadmium. Using normal adult human whole blood hemolysates, cadmium was found to activate normal erythrocytic ALAD and to reverse the in vitro lead-induced inhibition of ALAD at concentrations 40- to 100-fold lower than those of zinc. At higher concentrations cadmium was observed to inhibit erythrocytic ALAD activity, with a 50% inhibition of ALAD activity occurring at a concentration approximately 20 times higher than that of lead. The inhibition caused by cadmium, unlike that caused by lead, could not be reversed by an in vitro addition of zinc. The present results indicate that, with respect to concentration, cadmium is a more potent activator of normal adult human erythrocytic ALAD activity than is zinc. It is suggested that the role of cadmium and zinc in increasing ALAD activity and reversing lead induced inhibition of ALAD activity is a structural one related to binding of the metal to peripheral sulfhydryl groups.     

Effect of metal salts on UDPglucuronosyltransferase activity of various aglycones in rat liver microsomes in vitro

The effects of aluminium, manganese, ferrous and ferric iron, cobalt, nickel, copper, zinc, cadmium, barium, mercury and lead ions on the rat hepatic UDPglucuronosyltransferase activity with regard to 4-methylumbelliferone, 4-nitrophenol, bilirubin, and 2-aminophenol were investigated in vitro. Conjugation of bilirubin was found to be activated by manganese, cobalt, nickel, cadmium, mercury and lead ions in native microsomes, but not in digitonin-treated microsomes. Aluminum, ferrous iron, cadmium, barium and lead increased the conjugation of 4-nitrophenol in native microsomes and aluminium in digitonin-treated microsomes as well. Lead slightly activated 4-methylumbelliferone conjugation, whereas none of the metal salts studied were found to activate the conjugation of 2-aminophenol. Copper and zinc salts inhibited all conjugations. In digitonin-treated microsomes, the conjugation of 4-nitrophenol was inhibited by cadmium and mercury, that of 4-methylumbelliferone by nickel and lead as well. The sensitivity of 4-methylumbelliferone conjugation to inhibition by mercury was most pronounced in microsomes prepared from rats pretreated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.     

Bioaccessibility of essential and non-essential metals in commercial shellfish from Western Europe and Asia

Maximum acceptable concentrations of metals in food – based on total concentrations – have been established in many countries. To improve risk assessment, it would be better to take into account bioaccessible concentrations. A total of seven species of molluscs from France, UK and Hong Kong was examined in this study including clams, mussels, oysters, scallops and gastropods. The species which have total metal concentrations higher than the most severe food security limits are mainly oysters (all of the three studied species), the gastropod Buccinum undatum for cadmium and zinc, and scallops for cadmium. The lowest bioaccessibility (in % extractability with gut juices) was observed for silver (median for all of the species: 14%), it was moderate for lead (median: 33%) and higher for cadmium, zinc and copper (medians were respectively 54%, 65%, and 70%). In most cases, bioaccessible concentrations remained higher than the safety limits, except for cadmium in scallops and zinc in B. undatum. The influence of feeding habit (masticated or swallowed, addition of vinegar or lemon) on metal bioaccessibility in oysters is limited. On the contrary, cooking the gastropods decreased the bioaccessibility of metals, except silver.     

Disposition of metals in rats: a comparative study of fecal, urinary, and biliary excretion and tissue distribution of eighteen metals

Fecal (0-4 days), urinary (0-4 days), and biliary (0-2 hr) excretion and tissue distribution of 18 metals were examined in rats after iv administration. Total (fecal + urinary) excretion was relatively rapid (over 50% of dose in 4 days) for cobalt, silver, and manganese; was between 50 and 20% for copper, thallium, bismuth, lead, cesium, gold, zinc, mercury, selenium, and chromium; and was below 20% for arsenic, cadmium, iron methyl mercury, and tin. Feces was the predominant route of excretion for silver, manganese, copper, thallium, lead, zinc, cadmium, iron, and methyl mercury whereas urine was the predominant route of excretion for cobalt, cesium, gold, selenium, and chromium; while both excretion routes were equally important for bismuth, mercury, arsenic, and tin. Biliary excretion seems to be an important determinant for the fecal excretion of silver, arsenic, manganese, copper, selenium, cadmium, lead, bismuth, cobalt, and methyl mercury. Between 45 (silver) and 0.8% (methyl mercury) of the dosages administered of these metals was excreted into bile in 2 hr, and they exhibited high bile/plasma concentration ratios. The biliary excretion of copper, selenium, lead, and chromium did not increase proportionally with dosage, suggesting that the hepatobiliary transport of these metals is saturable. The fraction of dosage excreted into bile was independent of the dosage for silver, arsenic, manganese, bismuth, methyl mercury, mercury, gold, cesium, thallium, and tin, but markedly increased with increase in dosage of cadmium, cobalt, zinc, and iron. The latter phenomenon is probably due to saturation of hepatic (cadmium, zinc) or extrahepatic (iron) metal-binding sites. Comparison of biliary and fecal excretion rates indicates that arsenic and selenium undergo intestinal reabsorption, whereas thallium and zinc enter the feces also by non-biliary routes. Most of the metals reached the highest concentration in liver and kidney. However, there was no direct relationship between the distribution of metals to these excretory organs and their primary route of excretion.     

Synthesis and properties of the monomethyl ester of meso-dimercaptosuccinic acid and its chelates of lead (II), cadmium(II), and mercury(II).

The monomethyl ester of meso-dimercaptosuccinic acid (MoMeDMSA) and its chelates with lead(II), cadmium(II), and mercury(II) have been synthesized. The mercury(II) chelate of MoMeDMSA is formed by the coordination of the two sulfur atoms in MoMeDMSA, whereas the lead(II) and cadmium(II) chelates are formed by the coordination of one sulfur and one oxygen atom. The solubilities of the chelates are pH dependent; the mercury(II) chelate dissolves when the uncoordinated carboxylic acid dissociates, but the lead(II) and cadmium(II) chelates are solubilized only after the uncoordinated mercapto group is dissociated. The cadmium(II) chelate is dimeric and the lead(II) and mercury(II) chelates are monomeric at the concentrations and conditions used in this study. The acid dissociation constants of the chelating agent and the uncoordinated groups in its metal chelates have been determined in 50% v/v methanol-water. These acid-base properties in addition to the polarity of the chelating agent contribute to the effectiveness in the in vivo mobilization of intracellular in vivo deposits of cadmium. The biliary excretion of cadmium in rats increased by a factor of 173 upon administration of the relatively toxic, nonpolar dimethyl ester of DMSA whereas the administration of the less toxic, more polar monomethyl ester increased the biliary excretion of cadmium by a factor of 63. On the other hand, meso-DMSA which is highly polar and less toxic is known to be without effect on biliary excretion of cadmium. The monomethyl DMSA, therefore, appears to have properties that are intermediate between those of DMSA and its dimethyl ester, as far as both chelating properties and biliary excretion of cadmium are concerned.     

δ-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*

Abstract: The mechanisms underlying mouse δ-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 μM 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 μM EDTA or 500 μM 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 μM). 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 Znβ previously described in mammalian and bacterial ALA-D.     

The effects of heavy metals on [3H]thymidine uptake in lymphocytes.

The effects of lead, cadmium, mercury, and zinc were determined on the response of mouse splenic lymphocytes cell cultures to stimulation by the mitogens phytohemagglutinin (PHA) and pokeweed mitogen (PWM). Mice were exposed for 30 days to the metal salt in the drinking water. Splenic lymphocytes were cultured at 15 and 30 days to determine the response to the mitogens by measuring the uptake of [³H]thymidine. In vitro response was measured after adding the metal salts directly to the cell culture. In vivo exposure to high concentrations of lead, cadmium, or mercury for 30 days resulted in significantly (p < 0.05) decreased responses to both mitogens. Cadmium (1.42 mm) and mercury (0.50 mm) produced these decreases after 15 days of exposure. Cadmium and mercury also produced dose-dependent inhibition in vitro, with the response to PHA being more sensitive. The results suggest that the metals may affect the lymphocyte directly by altering the synthesis of cellular DNA and thereby influence production of antibodies. Lymphocyte membrane ATPase activity was measured to ascertain the metal effects. All metals used caused a dose-dependent inhibition of ouabain-insensitive ATPase in isolated lymphocytes.     

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.     

Stress proteins induced by exposure to sublethal levels of heavy metals in sea bream (Sparus sarba) blood cells.

Blood cells freshly collected from silver sea bream (Sparus sarba) were exposed in vitro to different sublethal concentrations of cadmium(II), lead(II) or chromium(VI). HSP70 stress proteins were significantly overexpressed after exposure to metal concentration as low as 0.1 microM. Under our experimental conditions, no overexpression of metallothioneins in blood cells was evidenced. Our results show that fish blood cells may constitute an interesting biological model for experimental and applied toxicology, especially in the case of environmental pollution.     

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.     

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.     

Subject index for volume 57

Non-selenium glutathione peroxidase was isolated from calf liver to approximately 70% of purity (by analytical and sodiumdodecyl sulfate-gel electrophoresis) by a simple purification procedure. The preparation had a specific activity of 74 units/mg with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate and 14 units/mg with cumene hydroperoxide (COP). Both the peroxidase and transferase activities of the enzyme were markedly inhibited by cadmium, methylmercury, mercury, and lead. Of the various metal ions tested, cadmium was the most potent inhibitor. The nature of the inhibition of the transferase activity by metal ions was noncompetitive with respect to glutathione and CDNB. The peroxidase activity also was inhibited noncompetitively with respect to glutathione and COP. The inhibition constants (K_i) for cadmium, methylmercury, mercury, and lead were calculated to be 2, 4, 6.5, and 9 × 10^?5m, respectively, for the transferase activity and 1.5, 7, 9, and 3 × 10^?6m, respectively, for the peroxidase activity. These findings suggest that metal ion-induced lipid peroxidation may be mediated in part via inhibition of non-selenium glutathione peroxidase activity. In addition, metal ion toxicity may be expressed in part through inhibition of glutathione transferase activity which plays an important role in the detoxification of a broad spectrum of compounds.     

Effect of heavy metals on human rheumatoid synovial cell proliferation and collagen synthesis

The dose-dependent effects of heavy metals on cell proliferation, collagen synthesis, and non-collagen protein synthesis were studied in early passage cultures of human synovial cells exposed to 1-100 microM concentration of gold, silver, mercury, cadmium or lead for 5 days. The incorporation of [3H]thymidine into trichloroacetic acid insoluble material was inhibited 50% by each of the heavy metals at concentrations between 1 and 10 microM. Gold, lead and mercury (10 microM) decreased the DNA content of the cultures by less than 15%; silver (10 microM) and cadmium (10 microM) resulted in decreased DNA content, which was attributed to cytotoxicity. A dose-dependent inhibition of [3H]proline incorporation into bacterial collagenase resistant (non-collagen) protein was observed after incubation with 10 microM mercury, lead and silver. During incubations with 10 microM gold and cadmium, collagenase resistant protein accumulation increased. All the heavy metals except for gold inhibited collagen accumulation to a greater extent than non-collagen protein accumulation. Gold (10 microM) stimulated the amount of collagen produced per cell, and the percentage of collagen to total protein was increased 50%. The rate of collagen accumulation in medium decreased during incubation with 10 microM silver, mercury, cadmium and lead. The stimulation of collagen synthesis may be a unique property of gold related to the therapeutic indices of gold, compared to other heavy metals, in rheumatoid arthritis.     

Interrelationships of glutathione reductase, 5-aminolevulinic acid dehydratase, and free sulfhydryl groups in the erythrocytes of normal and lead-exposed persons

Blood lead, erythrocyte glutathione reductase (GSSG-R), 5-aminolevulinic acid dehydratase (ALA-D), and free sulfhydryl (SH) groups were measured in normal subjects and in those with occupational exposure to lead. With increasing blood lead concentration the activity of GSSG-R rises and that of ALA-D decreases. There is also a fall in the level of free SH with rising blood lead concentrations. There is a high degree of correlation between these parameters, and it is suggested that the changes represent part of a biological control mechanism to compensate for the reduction of available sulfhydryl groups by lead ions.     

Influence of urbanization of the western coast of the United Arab Emirates on trace metal content in muscle and liver of wild Red-spot emperor (Lethrinus lentjan).

We hypothesized that increased ambient concentrations of metals, as a consequence of escalating urbanization and industrialization of the Gulf region will respond in increased contamination of edible fish species. In this study, we report concentrations of chromium, manganese, cobalt, copper, zinc, arsenic, cadmium, mercury and lead in meat and liver of wild Red-spot emperor (Lethrinus lentjan) from three sampling points at the UAE coast. Analysis was performed by the ICP-MS/microwave digestion. Our study has shown that meat and liver metal content was significantly higher in areas with higher industrial activity, although metal values did not exceed permitted levels of fish for human consumption.     

In vitro effects of lead, mercury and cadmium on the enzymic activity of red-blood cell pyrimidine 5'-nucleotidase

The decrease of pyrimidine 5'-nucleotidase (P5N) in human erythrocytes, detected in persons occupationally exposed to lead (Pb), is not an in vitro artefact resulting from the release of lead during erythrocyte hemolysis but is a true reflection of the enzyme activity in vivo. Like Pb, mercury (Hg) and cadmium (Cd) ions exhibit an important in vitro inhibitory action on the P5N enzymic activity. However, when the metals are preincubated as metallothionein complexes with P5N preparations, their inhibitory power disappears.     

Relationship between lead concentration in blood and the activities of erythrocyte pyrimidine 5′-nucleotidase and delta-aminolevulinate dehydratase in the mice exposed to lead

The relationship between the activities of both pyrimidine 5-nucleotidase (Py5N) and delta-aminolevulinate dehydratase (ALA-D) in erythrocytes and the concentration of lead in blood was investigated in the mice which were given ad libitum a drinking water containing lead of 10 to 500 ppm, for 30 days.From these results, it was demonstrated that the erythrocyte PySN activity is inhibited by lead and its activity level is negatively correlated with the concentration of lead in blood (r=–0.78). In addition, it was suggested that the erythrocyte Py5N activity is a better indicator in the exposure to relatively high lead concentration while the ALA-D is a more sensitive indicator for evaluating the lead exposure of low and moderate levels.