Risk assessment of heavy metal toxicity by sensitive biomarker δ-aminolevulinic acid dehydratase (ALA-D) for onion plants cultivated in polluted areas in Kosovo.

Biomarkers allow an integrated risk assessment of heavy metal pollution effects in living organisms. In this study, the biochemical effects of Cd, Cr, Ni, Pb and Zn pollution in agricultural soil and their accumulation in Alium cepa L. (onion) were evaluated with ALA-D enzyme response as a biomarker, along with δ-aminolevulinic acid (ALA) and total chlorophyll contents in leaves of this plant. Soil samples were randomly selected from agricultural areas in two regions, Mitrovica and Obiliqi, which are considered the most industrially polluted regions in Kosovo. Results show that Pb and Zn concentrations in soil samples from Mitrovica (1953-2576 mg kg -1) and Obiliqi regions (138-179 mg kg -1) and their bioaccumulation levels in onion were significantly higher in comparison with the control group. There was an adverse negative correlation between Pb or Zn concentration and ALA-D activity and total chlorophyll content, and a positive correlation with ALA content. This study indicates that ALA-D activity can be used as a very sensitive biomarker for evaluation of heavy metal pollution. The bioaccumulation of heavy metals from soil polluted areas poses a threat for food contamination and public health.

Oral administration of diphenyl diselenide potentiates hepatotoxicity induced by carbon tetrachloride in rats.

Carbon tetrachloride (CCl4) is a model for studying free radical-induced liver injury and screening hepato-protective drugs. Numerous studies have reported the involvement of oxidative stress in CCl4-induced liver damage and the hepato-protective effects mediated by different antioxidants. The present study examined the effects of diphenyl diselenide, (PhSe)2, on hepatotoxicity induced by CCl4 in rats. To this end, male Wistar rats received (PhSe)2 by oral route at the dosage of 31.2 mg/kg for one or two days. After the second day of treatment, rats received CCl4 orally in a single dose. The liver and kidney were utilized for determination of histopathology, biochemical [aspartate (ALT) and alanine (AST) aminotransferases, alkaline phosphatase (ALP), total bilirrubin (TB) and gamaglutamyl transferase (GGT)] and toxicological parameters [thiobarbituric reactive species (TBARS) levels, catalase activity, ascorbic acid, nonprotein thiols (NPSH) and aminolevulinate dehydratase (-ALA-D) activity]. Repeated administration of (PhSe)2 caused a marked potentiation of hepatotoxicity induced by CCl4 exposure, as manifested by an increase in biochemical parameters (AST, ALT, ALP, GGT and BT) and severe alteration in histopathology. This study also demonstrated a potentiation of TBARS levels and a consequent depletion of important antioxidant defenses including catalase and ascorbic acid. Pre-treatment with a single dose of (PhSe)2 prevented the effect of strychnine, a substrate for CYPs, abolishing lethality in mice. This result indicates that (PhSe)2 prevented animal death, suggesting an activator action of (PhSe)2 in CYPs. This study clearly indicates that (PhSe)2 potentiated acute hepatic damage induced by CCl4.

Augmentation of hematoporphyrin uptake and in vitro-growth inhibition of L1210 leukemia cells by succinylacetone.

Succinylacetone (SA; 4,6-dioxoheptanoic acid), a specific inhibitor of delta-aminolevulinic acid dehydrase (ALAD) (the second enzyme of the heme biosynthetic pathway), was tested for its effect in L1210 cells from inbred DBA/2 mice. ALAD from broken L1210 cells was completely inhibited by 1 microM SA, but in whole cells activity was decreased only 83% after incubation of the cells with 2.5 mM SA for 3 days. When incubated with hematoporphyrin (HP), L1210 cells rapidly took up porphyrin from the medium, and this uptake could be augmented by pretreatment of the cells with SA; but this enhancement of porphyrin uptake occurred gradually over a period of days. When SA-treated and untreated L1210 cells were incubated with increasing concentrations of HP in the medium, SA-treated cells reached the saturation concentration of cellular porphyrin at lower medium HP concentrations than did untreated cells. Growth of L1210 cells could be inhibited by 2 mM SA or more. Addition of increasing amounts of serum to cultures of cells containing SA did not reverse the growth inhibition due to SA. Porphyrin uptake from HP in the medium in nonmalignant fibroblast line 3T3 was much lower than in L1210 cells and could not be enhanced by incubation of the cells with SA.

Two-dimensional gel electrophoretic analysis of cytoplasmic proteins from Friend erythroleukemia cells chemically induced to undergo terminal erythroid differentiation.

The proerythroblastoid Friend erythroleukemia cell (FELC) line, clone TR 19-9, was treated with 4 mM hexamethylene bisacetamide (HMBA) over a 6-day period. Greater than 94% of the FELC reacted positively to the benzidine assay for hemoglobin by Day 4 of treatment. Protein accumulation during the final 4 days of treatment (from Days 2 to 6) was monitored by labeling for 24-h periods with a 14C-labeled amino acid mixture. At the end of each radiolabeling time point, cells were harvested and cytoplasmic proteins were isolated and subjected to two-dimensional gel electrophoresis in triplicate. Short-term fluorographic exposures were made in the linear X-ray film response range to monitor those polypeptides which were most rapidly accumulated. Fluorographs were digitized for computer image analysis and gel data comparison rationales were used to combine the polypeptides contained on the replicate fluorographs into a single cytoplasmic polypeptide profile or Master Image for each of the two experimental conditions, control and HMBA-treated FELC. These two images were merged into a single Master Composite Image containing a total of 211 polypeptides so that those polypeptides common to both and/or unique to each of the experimental conditions could be viewed graphically in the same plane. A total of 98 polypeptides in HMBA-treated FELC were shown to have large accumulation rate differences from the control FELC;32 of these polypeptides were present in the HMBA Master Image which were not detected in the Control Master Image and 66 polypeptides were present in the Control Master Image but not detected in the HMBA Master Image. Five polypeptides, found in both Master Images, were shown to vary quantitatively in the HMBA-treated FELC from the corresponding polypeptides in the control. These quantitative data measurements on the rates of accumulation of various common polypeptides offer a mode for simultaneously monitoring the kinetics of induction and repression of many gene products throughout an experimental time course.

Tissue distribution of succinylacetone in the rat in vivo: a possible basis for neurotoxicity in hereditary infantile tyrosinemia.

Neurologic dysfunction is a significant component of hereditary infantile tyrosinemia, an autosomal recessive disorder of man. The specific enzyme defect leads to endogenous production of the biochemical marker compound, succinylacetone (SA). Earlier study of the role which SA plays in generation of the renal Fanconi syndrome, also associated with this disorder, led to speculation that SA might also have neurotoxic effects. Thus, we have studied the distribution and impact on heme metabolism of SA in brain, liver and kidney from rats treated in vivo. Our results show far greater retention of SA in brain and kidney than in liver, by a ratio of approx. 3:1. Delta-aminolevulinate dehydratase (ALAD) was reduced to less than 10% of control activity in all three tissues after three daily injections; after a 7-day recovery, activity was regained at different rates in the three tissues. Total heme content of each tissue showed a steady decline beyond the treatment period, the most marked reduction being found in kidney. Porphyrin intermediates, heme oxygenase activity and cytochrome P-450 content evidenced varying responses to SA exposure which differed from tissue to tissue. Our results show that brain tissue sequesters SA and that heme biosynthesis in brain, as distinct from liver and kidney, is adversely affected. Such effects could result in impaired oxidative metabolism in brain, producing the CNS manifestations of tyrosinemia.

Efficacy of 2,3-dimercapto-1-propanesulfonic acid (DMPS) and diphenyl diselenide on cadmium induced testicular damage in mice.

The deleterious effect of acute cadmium-intoxication in mice testes was evaluated. Animals received a single dose of CdCl2 (2.5 or 5 mg/kg, intraperitoneally) and a number of toxicological parameters in mice testes were examined, such as delta-aminolevulinic acid dehydratase (delta-ALA-D) activity, lipid peroxidation, hemoglobin and ascorbic acid contents. Furthermore, the parameters that indicate tissue damage such as plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were also determined. Thus, a possible protective effect of 2,3-dimercapto-1-propane-sulfonic acid (DMPS) and diphenyl diselenide (PhSe)2 were studied. The results demonstrated an inhibition of delta-ALA-D activity, a reduction of ascorbic acid and an increase of lipid peroxidation induced by cadmium, indicating testes damage. Furthermore, we observed an increase of plasma LDH, AST and ALT activities. DMPS (400 mol/kg) and (PhSe)2 (100 micromol/kg) partially protected from the inhibitory effect of 2.5 mg/kg CdCl2 on delta-ALA-D and from the increase of TBARS (thiobarbituric acid reactive species) levels. (PhSe)2 therapy was effective in ameliorate ascorbic acid content when the cadmium dose was 2.5 mg/kg. Treatment with DMPS and (PhSe)2, individually or combined, was inefficient in reducing cadmium-induced plasma LDH and ALT activity increase. The use of combined therapy (DMPS plus (PhSe)2) proved to be efficient in decreasing cadmium levels in testes and in ameliorating plasma AST activity from animals that received the highest dose of cadmium.

Cloning and sequencing of a previously unidentified gene that is involved in the biosynthesis of heme in Escherichia coli.

We have isolated a number of porphyrin (Por)-synthesis mutants as light-resistant revertants of a light-sensitive strain delta visA (hemH) of Escherichia coli that accumulates protoPor IX in the cell. Among such mutants, we found a double mutant (H103) with mutations in hemA and in a new gene downstream of hemA. This new gene, designated hemK, was located at 27 min on the linkage map of the E. coli chromosome. By nucleotide (nt) sequencing, it was demonstrated that hemK forms part of the hemA-prfA-hemK operon and encodes 225 amino acids that show no significant homology to any protein in the standard databases. The mutant strain H103 formed small colonies and showed no catalase activity even in the presence of 5-aminolevulinic acid (ALA), indicating its inability to catalyze a step in the biosynthesis of heme from ALA. An extract of H103 cells has readily detectable ALA dehydratase and porphobilinogen deaminase activities. H103 cells carrying a plasmid that included only hemA as an insert accumulated protoPor and coproPor, but showed no sensitivity to light, a result that suggests that it may be deficient in protoporphyrinogen oxidase activity.

Succinylacetone and delta-aminolevulinic acid dehydratase in hereditary tyrosinemia: immunochemical study of the enzyme.

Immunochemical determinations of delta-aminolevulinic acid (ALA) dehydratase were performed in erythrocytes and in liver of a patient with hereditary tyrosinemia who underwent liver transplantation for correction of this metabolic disorder. Both erythrocytic and hepatic ALA dehydratase activities were extremely low before liver transplantation, but they appeared normal after transplantation. According to results of immunochemical quantification of ALA dehydratase, the level of the enzyme protein in erythrocytes was not different before, during, and after liver transplantation. Immunoquantifiable enzyme concentrations were not substantially different in the patient's own liver as compared with the transplanted liver. These findings indicate that although succinylacetone, an abnormal metabolite produced in tyrosinemia, is a potent inhibitor of the activity of ALA dehydratase, it has a far less effect on the synthesis of the enzyme protein.

Gasoline sniffing and tetraethyl lead poisoning in children.

Two cases of acute organic lead poisoning following gasoline sniffing, with one death, are reported. Signs of lead encephalopathy with elevated blood lead levels and markedly decreased levels of erythrocytic delta-amino levulinic dehydratase (ALAD) were demonstrated. Erythrocytic ALAD activity was used as a screening test for the detection of tetraethyl lead (TEL) poisoning in a group of 43 children who were presumed gasoline sniffers. Their mean ALAD activity was 190 units compared to a mean of 538 units for a control group. A survey of schoolchildren in another isolated community revealed that 59% had decreased ALAD activity, which correlated well with a history of gasoline sniffing. Only 5% of the children had blood lead levels over 40 microgram/dl. The two surveys herein reported suggest that there may be large numbers of children living in isolated communities who are suffering from TEL poisoning as a result of gasoline sniffing. This constitutes a major medical, public health, and social problem heretofore virtually unrecognized.

Enhancement of aminolevulinic acid based photodynamic therapy by adriamycin.

This paper reports on studies that evaluate the interaction between delta-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) and adriamycin (ADM) in an animal model system. Two groups of mice bearing a transplantable mammary adenocarcinoma received ADM i.p. in a single dose of 5 mg (low dose) and 30 mg (high dose) per kg body weight. Sixteen or 40 h after administration of the drug, mice were sacrificed, tumours, livers and hearts were removed and porphyrins, enzyme activities and malondialdehyde content were determined. Tumour explants of ADM-treated mice were incubated with ALA and irradiated with an He-Ne laser. Re-implantation of these in vitro PDT-treated explants into test animals showed that inhibition of tumour growth was significantly enhanced by combined treatment when the low dose of ADM was used. There were no significant changes in porphyrin content, ALA dehydratase and porphobilinogenase activities in the tissues analyzed after ADM treatment as compared with control values. ADM toxicity is thought to be related to semiquinone free radical formation with subsequent generation of reactive oxygen species such as peroxide and hydroxyl radical. These species are considered to initiate lipid peroxidation (LPO) and cause DNA damage. In the case of low-dose treatment with ADM a significant increase in the LPO product, malondialdehyde, was observed after PDT whereas with the high-dose regimen no changes were observed. In the case of explants of (non-irradiated) cardiac tissue malondialdehyde production was also found to be dependent on the dose and time of administration of adriamycin. In our in vivo/in vitro model system we have shown that pre-treatment with ADM increased the cytotoxicity of ALA-PDT at a dosage level of ADM which did not raise LPO levels in heart tissue. The mechanism of this effect has not been clearly elucidated but our data suggest that the observed enhancement of PDT may be attributed in part to the weakening of cellular defence mechanisms by the pre-treatment involving free radical generation by ADM.

Enzymes for heme biosynthesis are found in both the mitochondrion and plastid of the malaria parasite Plasmodium falciparum.

All eight enzymes required for de novo heme biosynthesis have been predicted from the nuclear genome of the human malaria parasite Plasmodium falciparum. We have studied the subcellular localization of three of these using a GFP reporter in live transfected parasites. The first enzyme in the pathway delta-aminolevulinic acid synthase (ALAS) is targeted to the mitochondrion, but the next two enzymes porphobilinogen synthase (PBGS) and hydroxymethylbilane synthase (HMBS) are targeted to the plastid. An enzymatically active recombinant version of PBGS from P. falciparum was over-expressed and its activity found to be stimulated by Mg2+ (and enhanced by Mn2+) but not by Zn2+. A hypothetical scheme for the exchange of intermediates in heme biosynthesis between the mitochondrion and plastid organelle, as well as organelle attachment is discussed.

Characterization of heme oxygenase activity in Leydig and Sertoli cells of the rat testes. Differential distribution of activity and response to cadmium.

Leydig and Sertoli cells of the rat testes differ with respect to the activities of the enzymes of the heme and hemoprotein degradative pathway and in their responses to Cd2+ treatment. The microsomal heme oxygenase activity in the Leydig cell preparations was nearly 9- to 10-fold greater than in Sertoli cell preparations, but the characteristics of the enzyme appeared to be similar in both cell populations, as judged by the cofactor requirements and the inhibitory action of heme ligands. Differences between the two cell preparations also were detected in the activity of NADPH-cytochrome c (P-450) reductase and in the contents of cytochrome P-450 and heme, with Leydig cells possessing the higher values. The activities of the cytosolic biliverdin reductase were comparable in both cell preparations. The significantly higher levels of porphyrins and the activities of delta- aminoleuvinate synthetase and uroporphyrinogen-I synthetase suggest that Leydig cells constitute the primary site of heme and hemoprotein biosynthetic activities. The mode of regulation of heme oxygenase activity in the testes and in the liver was compared. The responses of heme oxygenase to Cd2+ treatment (20 mumoles/kg, 24 hr) in the two testicular cell populations were dissimilar and both differed from that of the liver. In Leydig cells, heme oxygenase activity was decreased dramatically, whereas in the liver the activity was greatly increased. Heme oxygenase activity in Sertoli cells was refractory to Cd2+. The Cd2+-mediated decrease in heme oxygenase activity in Leydig cells did not reflect a direct inhibitory action of Cd2+ on the enzyme or a decreased total content of the microsomal protein. The dissimilarity between the mode of regulation of heme metabolic activities in the testes, when determined in Leydig cells, and that in the liver involved the inability of bromobenzene to evoke an increase in heme oxygenase activity and the age-related changes in the activities of heme oxygenase and delta- aminoleuvinate synthetase. In contrast to heme oxygenase activity, the heme concentration in Sertoli cells was remarkably sensitive to Cd2+ treatment, where a 7-fold increase in heme concentration was observed. The same treatment caused only a 2-fold increase in heme concentration in Leydig cells. In the latter cells, however, the increase in heme concentration was accompanied by a marked reduction in cytochrome P-450 levels. The cytochrome could not be measured in Sertoli cell preparations.(ABSTRACT TRUNCATED AT 400 WORDS)

Depletion of liver regulatory heme in benzene exposed rats.

The effect of a single dose of benzene (0.5 ml/kg body wt i.p.) on the heme saturation of tryptophan pyrrolase activity in liver was examined. There was a significant decrease in the heme saturation of hepatic tryptophan pyrrolase, suggesting depletion of "regulatory heme". After benzene administration there was significant increase in delta-aminolevulinate (ALA) synthetase activity (approx. 2-fold) while delta-aminolevulinate dehydratase activity was significantly decreased, however, ferrochelatase and heme oxygenase activities were unaltered. Administration of tryptophan to benzene pretreated rats showed a reversal of benzene effects on heme synthesizing enzymes: there is an increase in the heme saturation of tryptophan pyrrolase and decrease in delta-aminolevulinate synthetase. However, there was no significant alteration in the activity of delta-aminolevulinate dehydratase.

Study of erythrocyte delta-aminolevulinic acid dehydratase activity in porphyria cutanea tarda.

The erythrocyte delta-aminolevulinic acid dehydratase activity was studied in porphyria cutanea tarda patients, compared to healthy controls, in an attempt to resolve the contradictions in the relevant literature data. In an in vitro experimental system, a study was also made of how the erythrocyte delta-aminolevulinic acid dehydratase activity varies on the action of the activators -SH and Zn2+. It was found that, compared to the healthy controls, the erythrocyte delta-aminolevulinic acid dehydratase activity of porphyria cutanea tarda patients is significantly decreased, but it is restored to the original activity level on the addition of -SH and Zn2+. Since there is a general -SH requirement of delta-aminolevulinic acid dehydratase, the most obvious explanation for the decrease of the activity in the case of the patients is the shift of the natural redox systems of the erythrocytes, and the decrease of the reduced glutathione/oxidized glutathione ratio.

Biological availability of lead in a paint aerosol. 2. Absorption, distribution and excretion of intratracheally instilled lead paint particles in the rat.

Four groups of rats received by intratracheal instillation (1) a lead chromate paint particulate suspension, (2) lead tetraoxide suspension, (3) lead acetate solution, or (4) saline. Lead-dosed animals received an equivalent dose of 1 mg lead/kg body weight. Distribution of lead was monitored through assays of urine, feces, and tissues (lung, bone, muscle kidney, liver) obtained at post-mortem 5 weeks after exposure. Delta-aminolevulinic acid dehydratase (ALA-D) activity was measured to determine the effect of lead on heme biosynthesis. The vast majority of the dosed lead in the paint matrix remained in the lung. In contrast, in the lead acetate-dosed animals, little remained in the lung, but significant elevations were found in bone and kidney. Blood ADA-D was significantly depressed in the lead acetate-treated animals, but was not significantly different from control animals in the animals dosed with lead paint or lead tetraoxide. These findings suggest that lead chromate in an alkyd resin paint matrix is poorly absorbed from the lung compared with lead acetate and lead tetraoxide.

Cadmium induced testicular damage and its response to administration of succimer and diphenyl diselenide in mice.

Acute effects of cadmium in mice testes were evaluated. Animals received a single dose of CdCl2 (2.5 mg/kg or 5 mg/kg, intraperitoneally) and a number of toxicological parameters in mice testes were examined such as delta-aminolevulinic acid dehydratase (delta-ALA-D) activity, lipid peroxidation, hemoglobin content and components of the antioxidant defenses (superoxide dismutase (SOD) activity and ascorbic acid concentration). Furthermore, a possible protective effect of meso-2,3-dimercaptosuccinic acid (DMSA) and diphenyl diselenide (PhSe)2 are studied. The results demonstrated inhibition of delta-ALA-D and SOD activities, reduction in ascorbic acid, increase of lipid peroxidation induced by cadmium, indicating testes damage. DMSA (400 micromol/Kg) and (PhSe)2 (100 micromol/Kg) protected inhibitory effect of 2.5 mg/kg CdCl2 on delta-ALA-D and restored the increase of TBARS levels. Otherwise, (PhSe)2 treatment was effective in reducing the increase of TBARS levels induced by 5 mg/kg CdCl2, whereas DMSA and (PhSe)2, in combination, were ineffective in reducing TBARS level. However, these compounds alone or in combination, were unable to protect SOD activity and to improve ascorbic acid levels near to the normal value. The use of combined therapy (DMSA plus (PhSe)2) not proved be better than the monotherapy, in improving toxicological parameters evaluated in this model of testicular damage induced by cadmium.

Sensitivity of delta-ALA-D (E.C. 4.2.1.24) of rats to metals in vitro depends on the stage of postnatal growth and tissue.

Heavy metals, like cadmium, lead, and mercury, are potential toxic substances. The exposure to these metals can cause renal disturbances and neurological alterations. Young rats are more sensitive to harmful agents than adult animals. Delta-ALA-D enzyme acts as a biomarker of these exposures, since it has high affinity for divalent metals. The purpose of this search was to investigate the sensitivity of delta-ALA-D from suckling rats to cadmium, lead or mercury in vitro. IC(50) for delta-ALA-D activity of brain, kidneys, and liver from rats with ages between 1 and 6, 8 and 13 or 17 and 21 days was determined using metals concentrations that range from 0 to 200 microM for CdCl(2), 0 to 600 microM for HgCl(2) and from 0 to 50 microM for lead acetate. The results demonstrated that the cerebral delta-ALA-D activity is more sensitive to lead acetate than to cadmium and mercury. Delta-ALA-D from hepatic tissue is the most resistant to presence of mercury chloride in assay medium. Lead and cadmium are more toxic to renal enzyme than mercury. To sum up, the sensitivity of delta-ALA-D enzyme of young rats to heavy metals studied depends on the phase of development and tissue.

Measurement of enzymes in environmental intoxications.

The paper details the enzymes which have been shown to be affected by certain zenobiotics, principally industrial pollutants, toxic metals, toxic gases and food additives. The role of mixed function oxidases is discussed and methods of assessing their activity indicated.

S-adenosyl-L-methionine: its effect on aminolevulinate dehydratase and glutathione in acute ethanol intoxication.

The effect of disulfiram and S-adenosyl-L-methionine (SAM) administration to acute ethanol intoxicated mice on the hepatic glutathione (GSH) concentration and aminolevulinic and dehydratase (ALA-D) activity was investigated. It was found that both GSH levels and ALA-D activity were decreased, and evidence suggested that the toxic action of ethanol was due to its conversion into acetaldehyde. Administration of SAM reverses the effects of acute alcohol abuse by increasing liver GSH availability. In vitro, hepatic ALA-D activity was not modified by ethanol; instead it was non-competitively inhibited by acetaldehyde. This inhibition was efficiently reversed by GSH and cysteine (CySH). Therefore, a mechanism for the action of ethanol on ALA-D, based on the inhibitory effect of acetaldehyde, is proposed.

Disturbances in heme biosynthesis in rabbits after administration per os of low doses of tin or lead.

The experiment was performed on female rabbits that received per os equimolar doses (17 microM Me/kg) of SnCl2 x 2 H2O or Pb (CH3 COO)2 every day for 5 d. The activity of delta-aminolevulinic acid dehydratase (ALA-D) in the whole blood, liver, kidneys, brain, spleen, and bone marrow, concentration of free erythrocyte protoporphyrins (FEP), activity of delta-aminolevulinic acid synthetase (ALA-S) in the liver and bone marrow, urine delta-aminolevulinic acid (ALA-U), and coproporphyrins (CP-U) were determined. Lead and tin concentrations in the blood were estimated. Lead caused a significant inhibition of ALA-D in the blood, increased FEP concentration, and ALA and CP excretion in urine of rabbits. Lead also decreased ALA-D activity in the bone marrow and in the liver, and did not change ALA-S activity in the liver and bone marrow. Tin did not change any of the examined indices. Tin doses applied in the present study, maintained within the limits of permissible standards of metal levels in human diet, did not affect the process of heme biosynthesis in rabbits.