Co-administration of meso 2,3-dimercaptosuccinic acid monoesters reduces arsenic concentration and oxidative stress in gallium arsenide exposed rats.

1. Gallium arsenide (GaAs), a semiconductor, exerts toxicity as a result of its constitutive moieties; that is, gallium and arsenic that becomes dissociated after exposure. The present study focuses on reducing arsenic concentration from the target organs using monoesters of meso 2,3-dimercaptosuccinic acid (DMSA) either individually or in combination. 2. Animals were exposed to GaAs (0.0014 mol/kg, orally for 8 weeks) and then treated with monoisoamyl DMSA (MiADMSA), monocyclohexyl DMSA (MchDMSA) or monomethyl DMSA (MmDMSA) either individually (0.3 mmol/kg, orally) or in combination (0.15 mmol/kg each, orally) for five consecutive days. 3. GaAs exposure significantly inhibited blood δ-aminolevulinic acid dehydrogenase (ALAD), suggesting alterations in the heme synthesis pathway. Whereas a significant increase in blood, liver and kidney reactive oxygen species accompanied by an increase in lipid peroxidation points to the involvement of oxidative stress in GaAs toxicity. 4. GaAs also significantly disturbed glutathione metabolism. Hepatic and renal catalase activity decreased significantly, whereas hepatic and renal superoxide dismutase activity, as well as serum transaminases activity, showed marginal increase. Treatment with MiADMSA in combination with MchDMSA showed better therapeutic efficacy compared with other treatments in the aforementioned variables. 5. Co-administration of MiADMSA with MchDMSA provided better therapeutic effects, including reduction of arsenic burden, compared with all other treatments.

A comparison of 5-aminolaevulinic acid- and its heptyl ester: dark cytotoxicity and protoporphyrin IX synthesis in human adenocarcinoma WiDr cells and in athymic nude mice healthy skin.

5-aminolevulinic acid heptyl ester was investigated in human adenocarcinoma WiDr cells and in healthy skin of athymic nude mice in comparison with 5-aminolevulinic acid (ALA). Incubation of WiDr cells with ALA and ALA heptyl ester resulted in production of protoporphyrin IX (PpIX). Concentrations higher than 0.01 mm of ALA heptyl ester and higher than 1 mm of ALA were cytotoxic. The dark cytotoxicity was not related to PpIX. Intracellular localization, photocytoxicity and photobleaching rate of PpIX were the same for both drugs, although a 100 times lower concentration of ALA heptyl ester (0.01 mm) was needed in comparison with ALA (1 mm) to induce the same level of PpIX. ALA heptyl ester, topically (but not systemically) applied, is a promising candidate for fluorescence diagnosis and photodynamic therapy. Special attention must be focused on the concentrations of ALA heptyl ester; as excess may lead to cytotoxicity and inefficient PpIX generation.

Estresse oxidativo em porfiria hepâtica experimental disparadapor succinilacetona - urn inibidor da âcido 5-aminolevulinico desidratase / Oxidative stress in experimental hepatic porphyria triggered bysuccinylacetone — an inhibitor of 5-aminoluvulinic acid dehydratase

Para otimizar urn modelo experimental para o estudo do desbalanço redox em porfirias relacionadas ao acúmulo de ácido 5-aminolevulinico-(ALA), via inibição da ALA desidratase-(ALA-D), ratos foram tratados com o éster metílico de succinilacetona-(SAME), um catabólito da tirosina que inibe fortemente a ALA-D, mimetizando o estado metabólico observado nos portadores de porfirias e tirosinemias. Estabeleceram-se modelos de tratamento agudo por 36 e 18 h. No primeiro, os animais receberam 3 injeções de SAME (10, 40 ou 80 mg/kg, grupos All-IV). No segundo, os animais receberam 3 injeções de 40 mg/kg de SAME, ALA ou éster metílico de ALA (grupos BII-IV), ALA:SAME (30:10 mg/kg, grupo BV), ou 10 mg/kg SAME (grupo BVI). Paralelamente, avaliou-se se os sintomas neurológicos característicos das porfirias decorriam de danos oxidativos mitocondriais. Para isso, aplicou-se uma tecnologia óptica para medidas da difusão da depressão cortical que determinou a oxigenação e o estado redox do cit c em mitocôndrias do córtex cerebral de ratos submetidos ao tratamento crônico com ALA (40 mg/kg), SAME (10 e 40 mg/kg) e ALA:SAME (30:10 mg/kg), a cada 48 h, durante 30 dias. Tratamento agudo/36 h: Os níveis de ALA no plasma, fígado, cérebro e urina e o clearance renal do ALA aumentaram nos grupos tratados. A atividade de ALA-D e a coproporfirina urinaria reduziram. A marcação para proteínas carboniladas, ferro e ferritina aumentou no fígado e cérebro dos grupos tratados, especialmente no All. Os níveis de malondialdeído hepática aumentaram no grupo AIV. A razão GSH/GSH+GSSG e a atividade de GPx cerebrais aumentaram nos grupos AIV e AIII, respectivamente. Consistentemente com estes dados indicando um desbalanço oxidativo induzido pelo SAME, alterações mitocondriais e citosólicas ultraestruturais foram reveladas, especialmente no fígado./Tratamento agudo/18 h: Os níveis de ALA plasmáticos aumentaram nos grupos tratados, exceto em BIV. 0 grupo Bll mostrou aumento dos níveis hepáticos...

To optimize an experimental model for studying redox imbalance in porphyrias related to 5-aminolevulinic acid (ALA) accumulation through the inhibition of ALA dehydratase (ALA-D), rats were treated with methyl ester of succinylacetone (SAME), a tyrosine catabolite that strongly inhibits ALA-D, what mimics the metabolic state observed in patients suffering from porphyrias and tyrosinemias. Models of acute treatment were established during 36 and 18 h. In the first model, animals received 3 injections of SAME (10, 40 or 80 mg/kg, groups All-IV). In the second model, animals received 3 injections of 40 mg/kg SAME, ALA or methyl ester of ALA (groups BII-IV), ALA:SAME (30:10 mg/kg, group BV), or 10 mg/kg SAME (group BVI). Concomitantly, we evaluated if the neurologic symptoms characteristics of porphyrias were a consequence of the oxidative mitochondria! impairment. For this, an optical technology for the measurement of cortical spreading depression was applied. This techonology determined the cerebral oxygenation and the redox state of cit c in mitochondria of the cerebral cortex of rats submitted to a chronic treatment with ALA (40 mg/kg), SAME (10 and 40 mg/kg) and ALA:SAME (30:10 mg/kg), alternate days, during 30 days. Acute treatment/36 h: ALA levels in plasma, liver and urine and clearance of renal ALA increased in treated groups. ALA-D activities and urinary coproporphyrin were found to be decreased. Liver and brain proteins carbonyl, iron and ferritin were higher in the liver of treated groups, especially in All. Liver j malondialdehyde levels were higher in group AIV. Cerebral GSH/GSH+GSSG ratio and GPx activities increased in groups AIV and AIII, respectively. Consistently with these data indicating SAME-induced oxidative imbalance, mitochondrial and cytosolic ultrastructural changes were revealed, especially in the liver. Acute treatment/18 h: Plasma ALA levels increased in all treated groups but BIV. Group BII showed increased hepatic ALA levels…

The influence of melatonin and N-acetylcysteine in delta-aminolevulinic acid and lead induced genotoxicity in lymphocytes in vitro.

As is well known from earlier studies, the genotoxic effect of lead exposure was partly attributed to the formation of the highly reactive oxygen metabolites (ROMs) in the blood. However, lead ions have no ability to generate ROMs. Therefore, the recently published studies paid more attention to the role of delta-aminolevulinic acid (ALA) accumulation in lead-induced DNA damage. If the above-mentioned assumptions were taken into consideration, it seemed a reasonable approach to study the possible protective effects of antioxidants against genotoxic effects of lead. According to our results, N-acetylcysteine (NAC) and melatonin (MEL) were able to reduce significantly (p<0.05) the lead- and ALA-induced sister chromatid exchange frequencies in human lymphocytes in vitro. In spite of a relative reduction in the lead- and ALA-induced micronucleus formation in human lymphocytes, the reduction was not statistically significant (p>0.05). These results could be evaluated as supportive evidence for the hypothesis that increased antioxidant capacity of cells might fortify the efficiency of protective pathways against cytogenetic damage in lead exposure.

Effect of cadmium on chlorophyll biosynthesis and enzymes of nitrogen assimilation in greening maize leaf segments: role of 2-oxoglutarate.

Supply of cadmium chloride (0.5 mM) inhibited chlorophyll formation in greening maize leaf segments, while lower concentration of Cd (0.01 mM) slightly enhanced it. Inclusion of 2-oxoglutarate (2-OG, 0.1-10 mM) in the incubation mixture increased chlorophyll content in the absence as well as presence of Cd. Substantial inhibition of chlorophyll formation by Cd was observed at longer treatment both in the absence and presence of 2-OG. When the tissue was pre-incubated with 2-OG or Cd, the inhibition (%) of chlorophyll formation by Cd was lowered in the presence of 2-OG. Treatment with Cd inhibited ALAD activity and ALA formation and the inhibition (%) of ALA formation by Cd was strongly reduced in the presence of 2-OG. Glutamate dehydrogenase (GDH) activity was increased by the supply of Cd both in the absence as well as presence of 2-OG. In the presence of 2-OG, Cd supply significantly increased glutamate synthase (GOGAT) activity and reduced inhibition (%) of glutamine synthetase (GS) activity. The results suggested the involvement of the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway of ammonia assimilation to provide the precursor, glutamate, for ALA synthesis under Cd toxicity and 2-OG supplementation.

Mechanisms of 5-aminolevulinic acid ester uptake in mammalian cells.

The porphyrin precursor 5-aminolevulinic acid (ALA) is being widely used in photodynamic therapy of cancer. Improvement in ALA delivery has been sought through the use of ALA derivatives, in particular the esterification of ALA with aliphatic alcohols, which in certain cases can improve cellular penetration and selectivity. ALA uptake systems appear to be distinctive for each cell type. The LM3 mammary adenocarcinoma cell line takes ALA up by BETA transporters. In this work, we investigated ALA derivative transport systems through the inhibition of radiolabelled ALA uptake in the LM3 cells. We also performed inhibition studies of gamma-aminobutyric acid (GABA) uptake. The more lipohilic ALA derivatives hexyl-ALA and undecanoyl-ALA inhibit ALA uptake, whereas methyl-ALA, R, S-ALA-2-(hydroxymethyl)tetrahydropyranyl ester and the dendron aminomethane tris methyl 5-ALA does not inhibit ALA uptake. A similar pattern was found for GABA, except that the dendron inhibited GABA uptake. However, hexyl-ALA and undecanoyl-ALA are not taken up by BETA transporters, but by simple diffusion, although they still inhibit ALA uptake by binding to the cell membrane. These results show that different modifications to the ALA molecule lead to different uptake mechanisms. Whereas ALA is taken up by BETA transporters, none of the ALA derivatives shares the same mechanism. Knowledge of the mechanisms of ALA derivatives entry into the cells is essential to understand and improve ALA-mediated PDT and to the design of new ALA derivatives that may be taken up at a higher rate than ALA.

Dimethyl sulfoxide and ebselen prevent convulsions induced by 5-aminolevulinic acid.

We investigated whether intrastriatal (i.s.) administration of 5-aminolevulinic acid (ALA) induces oxidative damage and whether behavioral alterations induced by i.s. administration of ALA could be affected by antioxidants. Unilateral injection of ALA (6 micromol/striatum) increased (approximately 30%) thiobarbituric acid-reactive substances (TBARS), but did not affect striatal content of total thiol groups. ALA-induced body asymmetry was not prevented by pretreatment with ascorbic acid (100 mg/kg, s.c.), dimethyl sulfoxide (DMSO, 0.5 microl/striatum, i.s.) or ebselen (10 nmol/striatum, i.s.). ALA-induced convulsions were not prevented by ascorbic acid, but were partially prevented by DMSO and completely prevented by ebselen. Ebselen completely prevented the increase of striatal TBARS induced by ALA. Results obtained suggest the involvement of reactive species in ALA-induced convulsions and may be of value in understanding the physiopathology of neurological dysfunctions associated to ALA overload.

Melatonin prevents delta-aminolevulinic acid-induced oxidative DNA damage in the presence of Fe2+.

Delta-aminolevulinic acid (ALA), a heme precursor which accumulates during lead poisoning and acute intermittent porphyria, is reported to cause liver cancer. The carcinogenic mechanisms of ALA may relate to its ability to generate free radicals through metal-catalyzed oxidation which cause oxidative DNA damage. The aim of this study was to compare the efficacy of melatonin, trolox (vitamin E) and mannitol in altering DNA damage induced by ALA. Herein, we found, in the presence of Fe2+, that ALA-induced formation of 8-hydroxydeoxyguanosine in calf thymus DNA was dose and time-dependent. Melatonin, mannitol and trolox, all of which are free radical scavengers, inhibited the formation of 8-hydroxydeoxyguanosine in a concentration-dependent manner. The concentration of each (melatonin, mannitol and trolox) required to reduce DNA damage by 50%, i.e., the IC50, was 0.52, 0.84 and 0.90 mM, respectively.

Delta-aminolevulinic acid-induced lipid peroxidation in rat kidney and liver is attenuated by melatonin: an in vitro and in vivo study.

Acute intermittent porphyria (AIP) is a genetically inherited disease characterized by a partial block in liver heme biosynthesis and by increased urinary excretion of the delta-aminolevulinic acid (ALA). Recently, it has been proposed that the toxic effects of ALA are related to the generation of free radicals. In the present study the in vitro and in vivo effect of melatonin, a recently described antioxidative agent, on ALA-induced lipid peroxidation in rat liver and kidney was determined. The concentration of malonaldehyde (MDA) and 4-hydroxyalkenals (4-HDA) was assayed as an index of induced membrane oxidative damage. In vitro melatonin protected, in a concentration-dependent manner, against ALA-induced lipid peroxidation in liver and kidney homogenates. In in vivo experiments as well, it was demonstrated that ALA (40 mg/kg)-induced lipid peroxidation in liver and kidney was reduced by acute melatonin (10 mg/kg) treatment. The results support the involvement of free radicals in ALA toxicity and show that in vitro and in vivo melatonin confers protection against this toxicity, likely due to the antioxidative capability of the indole.

Melatonin protects against lipid peroxidation induced by delta-aminolevulinic acid in rat cerebellum, cortex and hippocampus.

The in vitro and in vivo effect of melatonin on delta-aminolevulinic acid-induced lipid peroxidation in rat cerebellum, cortex and hippocampus was determined. The concentration of malonaldehyde and 4-hydroxyalkenals was assayed as an index of induced membrane oxidative damage. The rise in malonaldehyde+4-hydroxyalkenals concentrations induced by delta-aminolevulinic acid in cerebellar homogenates was concentration-dependent (P < 0.001) and also time-dependent in cerebellar, cortical and hippocampal homogenates (P < 0.01). In vitro melatonin and vitamin E protected, in a concentration-dependent manner, against delta-aminolevulinic acid-induced lipid peroxidation in cortical, cerebellar and hippocampal homogenates. In in vivo experiments it was demonstrated that delta-aminolevulinic acid-induced lipid peroxidation (40 mg/kg) in cerebellum and hippocampus was reduced by acute melatonin (10 mg/kg) treatment (P < 0.05). The results show that both in vitro and in vivo melatonin confers protection against delta-aminolevulinic acid-induced oxidative toxicity in brain regions. The findings suggest that melatonin may be useful in reducing neural damage in individuals suffering from acute intermittent porphyria.

Modification of the photodynamic action of delta-aminolaevulinic acid (ALA) on rat pancreatoma cells by mitochondrial benzodiazepine receptor ligands.

We have shown that addition of exogenous delta-aminolaevulinic acid (ALA) to rat pancreatoma AR4-2J cells in culture leads to the increased production of porphobilinogen (PBG) and the accumulation of photoactive protoporphyrin IX (PPix) in these cells. Exposure to light (lambda > 400 nm) at an intensity of 0.2 mW cm-2 for 8 min resulted in an ALA dose-dependent cytolysis of the cells, with an EC50 of 6.6 +/- 0.7 microM. This cytolytic effect was light intensity dependent, with greater cell destruction after exposure to light at an intensity of 0.47 mW cm-2 than at 0.2 mW cm-2; it was also dependent on the duration of illumination, cell survival decreasing with increasing illumination times. The photodestruction of the AR4-2J cells following exposure to ALA can be attributed to the production of endogenous PPix, a photoactive porphyrin that we have shown to generate singlet oxygen upon illumination, whereas ALA itself does not. Further investigation of the molecular mechanisms underlying the photodynamic action of ALA demonstrated the involvement of the mitochondrial (peripheral) benzodiazepine receptor (MBR), a high-affinity recognition site for dicarboxylic porphyrins, and especially PPix. The centrally acting benzodiazepine compounds clonazepam and flumazenil, which have negligible affinities for the MBR, had no effect on ALA-mediated phototoxicity. In contrast, both the isoquinoline carboxamide PK11195 and the benzodiazepine Ro 5-4864 ligands, displaying a high affinity for the MBR, did affect ALA-mediated phototoxicity, each markedly increasing the EC50 for cell photodestruction and thus exerting a photoprotective effect. It is concluded that the MBR may play an important role in the expression of ALA-mediated PPix phototoxicity and that MBR ligands, by diminishing the actions of endogenous PPix, have the potential to rescue cells from porphyrin-induced photolysis.

Calcium-dependent mitochondrial oxidative damage promoted by 5-aminolevulinic acid.

Swelling of isolated rat liver mitochondria is shown to be induced by metal-catalyzed 5-aminolevulinic acid (ALA) aerobic oxidation, a putative endogenous source of reactive oxygen species (ROS), at concentrations as low as 50-100 microM. In this concentration range, ALA is estimated to occur in the liver of acute intermittent porphyria patients. Removal of Ca2+ (10 microM) from the suspension of isolated rat liver mitochondria by added EGTA abolishes both the ALA-induced transmembrane-potential collapse and mitochondrial swelling. Prevention of the ALA-induced swelling by addition of ruthenium red prior to mitochondrial energization by succinate demonstrates the deleterious involvement of internal Ca2+. Addition of MgCl2 at concentrations higher than 2.5 mM, prevents the ALA-induced mitochondrial swelling, transmembrane potential collapse and Ca2+ efflux. This indicates that Mg2+ protects against the mitochondrial damage promoted by ALA-generated ROS. The ALA-induced mitochondrial damage might be a key event in the liver mitochondrial damage of acute intermittent porphyria patients reported elsewhere.

A comparative study of the effects of delta-aminolaevulinic acid and the GABAA agonist, muscimol, in rat jejunal preparations.

Preparations of rat jejunum were tested for their responsiveness to the GABAA receptor agonist, muscimol, and to the haem precursor, delta-aminolaevulinic acid (ALA). Both muscimol (1.0-30 microM) and ALA (1.0 microM-3.0 mM) elicited a concentration-dependent increase in tone. Pretreatment with the GABAA antagonist, bicuculline (10(-5) M), blocked effects of muscimol at all concentrations tested and attenuated effects of 0.3 mM ALA. However, bicuculline enhanced responsiveness of the preparations to ALA at low concentrations (0.01-0.05 microM), as also did picrotoxin (10(-5) M), eliciting a significant increase of tone. The significance of these findings is discussed. This finding of pharmacological activity by ALA at concentrations comparable with its blood levels during acute attacks of intermittent porphyria provides support for the proposal that is may play a role in the aetiology of the gastrointestinal manifestations of this disease.

Tin(Sn+4)-diiododeuteroporphyrin; an in vitro and in vivo inhibitor of heme oxygenase with substantially reduced photoactive properties.

Iodination of Sn-deuteroporphyrin (Ki = 0.185 microM) at positions C2 and C4 of the porphyrin ring results in an enhanced ability of the resulting derivative, Sn-diiododeuteroporphyrin, to inhibit (Ki = 0.069 microM) heme oxygenase activity in vitro. The potency of Sn-diiododeuteroporphyrin inhibition of bilirubin production in vivo is similar to that of Sn-protoporphyrin, but in vitro tests demonstrate that, when in solution with human serum albumin, Sn-diiododeuteroporphyrin is significantly (3-10 times, depending upon conditions) less photosensitizing than are Sn-protoporphyrin or Sn-mesoporphyrin. These findings demonstrate that halogenation of a suitable porphyrin macrocycle can substantially diminish photoactive properties of the compound whereas retaining its ability to act as a heme oxygenase inhibitor.

Melatonin counteracts the 5-aminolevulinic acid-induced rise of rat forebrain tryptophan and serotonin concentrations at night.

When rats were exposed to extended light into the normal dark phase, the administration of 5-aminolevulinic acid (5-ALA) markedly stimulated the accumulation of tryptophan (TRP) and the synthesis of serotonin (5-HT) in the forebrain. When a single injection of melatonin (25 micrograms) was given near the end of the 5-ALA treatment, the rise in forebrain TRP levels was either totally prevented or reversed; likewise, the increased levels of 5-HT were also suppressed. Melatonin by itself also slightly suppressed forebrain TRP levels in non-5-ALA-treated rats. Whether these are pharmacological or physiological effects of melatonin remains to be determined.

Transformation of glutamate to delta-aminolevulinic acid by soluble extracts of Chlorobium vibrioforme.

Formation of the tetrapyrrole pigment precursor delta-aminolevulinic acid (ALA) from glutamate was detected and partially characterized in extracts of the strictly anaerobic green photosynthetic bacterial species Chlorobium vibrioforme by using assay methods derived from those developed for algae and cyanobacteria. ALA formation in Chlorobium extracts was saturated at 10 mM glutamate and required NADPH and ATP at optimal concentrations of 0.3 and 3 mM, respectively. Preincubation of the enzyme extract with RNase A destroyed the ALA-forming activity completely. Activity in the RNase-treated extract was restored by supplementation with Chlorobium RNA after addition of RNasin to block further RNase action. RNA from the cyanobacterium Synechocystis sp. strain PCC 6803 and Escherichia coli tRNAGlu also restored activity. Activity was inhibited 50% by 0.2 microM hemin. ALA formation was completely abolished by the addition of 5 microM 3-amino-2,3-dihydrobenzoic acid (gabaculine). These results indicate that Chlorobium extracts share with those of plants, eucaryotic algae, cyanobacteria, prochlorophytes, and methanogens the capacity for RNA-dependent ALA formation from glutamate.

Regulation of rat liver tryptophan pyrrolase by its cofactor haem: Experiments with haematin and 5-aminolaevulinate and comparison with the substrate and hormonal mechanisms.

1. The administration of haematin or 5-aminolaevulinate to rat enhances the activity of liver tryptophan pyrrolase; both endogenous and newly formed apoenzymes become strongly haem-saturated. Haem activation does not stabilize tryptophan pyrrolase. 2. Actinomycin D, puromycin or cycloheximide prevent the activation of the enzyme by 5-aminolaevulinate but not that by haematin. The latter is inhibited by haem-destroying porphyrogens. 3. The combined injection of either haematin or 5-aminolaevulinate with cortisol does not produce an additive effect, whereas potentation is observed when tryptophan is jointly given with either the cofactor or the haem precursor. 4. Further experiments on the substrate (tryptophan) mechanism of pyrrolase regulation are reported, and a comparison between this and the cofactor and hormonal mechanisms is made. 5. It is suggested that the substrate mechanism may also involve increased haem synthesis. 6. The role of tryptophan pyrrolase in the utilization of liver haem, and as a possible model for the exacerbation by drugs of human hepatic porphyrias, is discussed.