Tetracysteine-based fluorescent tags to study protein localization and trafficking in Plasmodium falciparum-infected erythrocytes.

UNLABELLED: Plasmodium falciparum (Pf) malaria parasites remodel host erythrocytes by placing membranous structures in the host cell cytoplasm and inserting proteins into the surrounding erythrocyte membranes. Dynamic imaging techniques with high spatial and temporal resolutions are required to study the trafficking pathways of proteins and the time courses of their delivery to the host erythrocyte membrane. METHODOLOGY AND FINDINGS: Using a tetracysteine (TC) motif tag and TC-binding biarsenical fluorophores (BAFs) including fluorescein arsenical hairpin (FlAsH) and resorufin arsenical hairpin (ReAsH), we detected knob-associated histidine-rich protein (KAHRP) constructs in Pf-parasitized erythrocytes and compared their fluorescence signals to those of GFP (green fluorescent protein)-tagged KAHRP. Rigorous treatment with BAL (2, 3 dimercaptopropanol; British anti-Lewisite) was required to reduce high background due to nonspecific BAF interactions with endogenous cysteine-rich proteins. After this background reduction, similar patterns of fluorescence were obtained from the TC- and GFP-tagged proteins. The fluorescence from FlAsH and ReAsH-labeled protein bleached at faster rates than the fluorescence from GFP-labeled protein. CONCLUSION: While TC/BAF labeling to Pf-infected erythrocytes is presently limited by high background signals, it may offer a useful complement or alternative to GFP labeling methods. Our observations are in agreement with the currently-accepted model of KAHRP movement through the cytoplasm, including transient association of KAHRP with Maurer's clefts before its incorporation into knobs in the host erythrocyte membrane.

Diphenyl diselenide and 2,3-dimercaptopropanol increase the PTZ-induced chemical seizure and mortality in mice.

The aim of the present study was to evaluate the interaction between a classic GABAergic antagonist -- pentylenetetrazol (PTZ) with an organoselenium compound -- diphenyl diselenide (PhSe)(2) and with the metal chelating agent -- 2,3 dimercaptopropanol (BAL). Mice were pre-treated with 150 micromol/kg (PhSe)(2) or BAL (250, 500 or 1000 micromol/kg) before treatment with PTZ. Pre-treatment with (PhSe)(2) reduced the latency for PTZ-induced seizure at doses of 40 and 60 mg/kg and cause a decrease in the latency for PTZ-induced death at the dose of 60 mg/kg. However, treatment with PTZ at dose of 80 mg/kg was not affected by (PhSe)(2) pre-treatment. Pre-treatment with BAL reduced the latency for PTZ-induced seizure at doses of 40 and 50 mg/kg. In addition, the latency for PTZ-induced death at the dose of 40 mg/kg was decreased significantly by pre-treatment with all doses of BAL. At the dose of 50mg/kg, a significant decrease in the latency for death occurred only in mice pre-treated with 500 and 1000 micromol/kg of BAL. Our results indicate that the PTZ-induced chemical seizures and mortality was enhanced by (PhSe)(2) and BAL. These results indicated that (PhSe)(2) and BAL interact with PTZ possibly by modulating the GABAergic system.

2,3-Dimercaptopropanol inhibits Ca2+ transport in microsomes from brain but not from fast-skeletal muscle.

Ca2+ is involved in the regulation of a variety of physiological processes, but a persistent increase in free cytosolic Ca2+ concentrations may contribute to cell injury. Dimercaprol (BAL) is a compound used in the treatment of mercury intoxication, but presents low therapeutic efficacy. The molecular mechanism responsible for the BAL toxicity is poorly known. In the present study, the effect of BAL and inorganic and organic mercury on Ca2+ transport by Ca2+-ATPases located in the sarco/endoplasmic reticulum of fast-skeletal muscle and brain was examined. Ca2+ uptake by brain and fast-skeletal muscle microsomes was inhibited in a dose-dependent manner by Hg2+. The calculated IC50 for Ca2+ uptake inhibition by HgCl2 was 1.05+/-0.09 microM (n = 8) for brain and 0.72+/-0.06 microM (n = 9) for muscle. The difference was significant at p < 0.01 (data expressed as mean +/- SD). At a low concentration (1 microM), 2,3-dimer-captopropanol had no effect on Ca2+ uptake by brain or muscle vesicles and did not abolish the inhibition caused by Hg2+. A high concentration of BAL (1 mM) nearly abolished the inhibition caused by 1.75 microM HgCl2 or 6 microM CH3HgCl in skeletal muscle. Surprisingly, at intermediate concentrations (40-100 microM) BAL partially inhibited Ca2+ transport in brain but had no effect on muscle. Furthermore, ATP hydrolysis by brain or muscle microsomes was not inhibited by BAL. These results suggest that in brain microsomes BAL affects in a different way Ca2+ transport and ATP hydrolysis. The increase in BAL concentration observed after toxic administration of this compound to experimental animals may contribute to deregulate Ca2+ homoeostasis and, consequently, to the neurotoxicity of BAL.

BAL modulates glutamate transport in synaptosomes and synaptic vesicles from rat brain.

The therapeutic use of BAL (2,3-dimercaptopropanol) as treatment for poisoning has been halted by data suggesting serious neurotoxicity. This article is a report on the effects of BAL and other dithiols, DMSA (meso-2,3-dimercaptosuccinic acid) and DMPS (2,3-dimercaptopropane-1-sulfonic acid), on [3H]glutamate release and uptake by rat brain synaptosomes and [3H]glutamate uptake by synaptic vesicles. BAL (100 microM) inhibited glutamate uptake (30%) and stimulated its basal release (30%) in synaptosomes, without affecting K+-stimulated release. BAL also inhibited glutamate uptake by synaptic vesicles (up to 60%). DMPS and DMSA (100 microM) had no significant effects on these parameters. The data reported here provide some evidence of glutamate involvement in BAL-induced neurotoxicity by demonstrating direct effects of BAL on glutamatergic system modulation.

Neonatal induction of tolerance to T(h)2-mediated autoimmunity in rats.

Brown-Norway (BN) rats are highly susceptible to drug-induced immune dysregulations and when injected with mercuric chloride (HgCl(2)) or sodium aurothiopropanolsulfonate (ATPS), they develop a syndrome characterized by a polyclonal B cell activation depending upon CD4(+) T(h)2 cells that recognize self-MHC class II molecules. Since peripheral tolerance of T(h)2 cells might be crucial in the prevention of immunological manifestations such as allergy, establishing conditions for inducing tolerance to HgCl(2)- or ATPS-mediated immune manifestations appeared to be of large interest. We report here that BN rats neonatally injected with HgCl(2): (i) do not develop the mercury disease, (ii) remain resistant to HgCl(2)-induced autoimmunity at 8 weeks of age and later, provided they are regularly exposed to HgCl(2), (iii) are still susceptible to ATPS-induced immune manifestations, and (iv) exhibit spleen cells that adoptively transfer tolerance to HgCl(2)-induced autoimmunity in naive, slightly irradiated, syngeneic recipients. These findings demonstrate that dominant specific tolerance can be neonatally induced using a chemical otherwise responsible for T(h)2-mediated autoimmunity.

Are we ready to replace dimercaprol (BAL) as an arsenic antidote?

1 Dimercaprol (BAL), 2,3-dimercaptopropanesulphonate sodium (DMPS) and meso-2,3-dimercaptosuccinic acid (DMSA) are effective arsenic antidotes, but the question which one is preferable for optimal therapy of arsenic poisoning is still open to discussion. Major drawbacks of BAL include (a) its low therapeutic index, (b) its tendency to redistribute arsenic to brain and testes, for example, (c) the need for (painful) intramuscular injection and (d) its unpleasant odour. 2 The newer antidotes DMPS and DMSA feature low toxicity and high therapeutic index. They can be given orally or intravenously due to their high water solubility. While these advantages make it likely that DMPS and DMSA will replace BAL for the treatment of chronic arsenic poisoning, acute intoxication-especially with lipophilic organoarsenicals-may pose a problem for the hydrophilic antidotes, because their ionic nature can adversely affect intracellular availability. 3 This article focuses on aspects dealing with the power of BAL, DMPS, and DMSA to mobilize tissue-bound arsenic in various experimental models, such as monolayers of MDCK (= Madin-Darby canine kidney) cells from dog kidney, isolated perfused liver from guinea-pigs, and perfused jejunal segments from rat small intestine. 4 The results show that hydrophilic DMPS and DMSA may fail to rapidly and completely remove arsenic that has escaped from the extracellular space across tight epithelial barriers. However, owing to their low toxicity, which allows larger doses to be applied, and the potential modification of their pharmacokinetics by means of inert oral anion-exchange resins, DMPS and DMSA may advantageously replace BAL whenever intervention time is not critical. With severe intoxication by organic arsenicals, when the point-of-no-return is a limiting factor, BAL may still have a place as an arsenic antidote.

Effect of mercuric chloride intoxication and dimercaprol treatment on delta-aminolevulinate dehydratase from brain, liver and kidney of adult mice.

Dimercaprol is a compound used in the treatment of mercury intoxication, however with low therapeutic efficacy. It is assumed that dimercaprol acts by reactivating target sulfhydryl-containing proteins. In the present investigation we studied the inhibitory effect of mercuric chloride treatment (3 days with 2.3 or 4.6 mg/kg HgCl2, sc) in mice on cerebral, renal and hepatic delta-aminolevulinate dehydratase (ALA-D) activity, and a possible reversal of the effect of mercury by dimercaprol (0.25 mmol/kg, 24 hr after the last mercury injection). Mercuric chloride did not inhibit cerebral ALA-D at the doses injected. Dimercaprol treatment did not restore the normal enzyme activity of the liver after the 25% inhibition caused by 4.6 mg/kg HgCl2. In the kidney, dimercaprol enhanced the inhibitory effect of 4.6 mg/kg mercuric chloride (from 35% after mercury treatment alone to 65% after mercury plus dimercaprol treatment). Mercury content increased in kidney after exposure to 2.3 or 4.6 mg/kg and the levels attained were higher than in any other organ Mercury accumulated in liver only after exposure to 4.6 mg/kg HgCl2, and dimercaprol further increased mercury deposition. Dimercaprol treatment also increased the levels of mercury in brain of animals exposed to 4.6 mg/kg HgCl2 The enzymes from all sources presented similar sensitivity to the combined effect of HgCl2 and dimercaprol in vitro. In the absence of preincubation, 0-500 muM dimercaprol potentiated the inhibitory effect of HgCl2 on ALA-D activity. In the presence of preincubation, and 100 and 250 muM dimercaprol enhanced ALA-D sensitivity to mercury, whereas 500 muM dimercaprol partially protected the enzyme from mercury inhibition. Dimercaprol (500 muM) inhibited renal and hepatic ALA-D when preincubated with the enzymes. These data suggested that the dimercaprol-Hg complex may have a more toxic effect on ALA-D activity than Hg2+. Furthermore, the present data show that dimercaprol did not acts by reactivating mercury-inhibited sulfhydryl-containing ALA-D, and that indeed it may have an inhibitory effect per se depending on the tissue.

Serum IgE concentration and other immune manifestations of treatment with gold salts are linked to the MHC and IL4 regions in the rat.

A subset of patients with rheumatoid arthritis occasionally develops skin reactions and glomerulonephritis and exhibits an increase in serum IgE concentration when treated with gold salts. Brown-Norway (BN) rats injected with aurothiopropanolsulfonate (ATPS) also manifest an autoimmune glomerulonephritis and increased serum IgE concentration, whereas Lewis (LEW) rats are resistant to complications. Here, we show linkage between responses to ATPS in a (BN x LEW) F2 cohort and the major histocompatibility complex (RT1) on rat chromosome 20 and between markers in the region of IL4 and other candidate genes on rat chromosome 10. Recently, human serum IgE concentration has been reported to be linked to the IL-4 region. Taken together, these findings raise the possibility that homologous genes could be implicated in ATPS manifestations in the rat and in the regulation of IgE levels in the human.

Genetic study of gold-salt-induced immune disorders in the rat.

BACKGROUND: Rheumatoid arthritis patients treated with gold salts occasionally develop a glomerulonephritis and an increase in serum IgE concentration. Brown-Norway (BN) rats injected with aurothiopropanolsulphonate (ATPS) exhibit an increase in serum IgE concentration, produce antilaminin antibodies (Abs) and develop glomerular linear immunoglobulin (Ig) deposits, occasionally a membranous glomerulopathy and vascular granular Ig deposits. Lewis (LEW) rats are resistant. METHODS: The genetic requirements governing the appearance of these manifestations were studied in congenic rats, and in F1 hybrids injected with ATPS. RESULTS: Non-MHC-linked genes from the BN strain were absolutely required for all the traits to be observed. The RT1n (BN) or RT1(1) (LEW) haplotypes at the MHC were permissive for all the manifestations to appear and two RT1(1) alleles were associated with the highest response. However, granular Ig deposits were only observed in RT1n rats. The high serum IgE concentration and the antilaminin Ab level were associated with the presence of glomerular Ig deposits but were not associated with the presence of vascular Ig deposits. CONCLUSIONS: This study shows that susceptibility to ATPS was mainly dependent upon non-MHC-linked BN genes and that the involvement of MHC-linked genes differed depending upon the character considered. There is an epistatic effect between the various genes.

Experimental gold-induced autoimmunity.

The pathogenesis of gold-induced autoimmunity and membranous glomerulopathy is not well understood. HgCl2 and D-penicillamine, other chemicals known to trigger membranous glomerulopathy in humans, induce autoimmune manifestations in Brown-Norway (BN) rats but not in Lewis (LEW) rats. These chemicals trigger T-cell clones which are specific for self class II molecules from the major histocompatibility complex and are probably responsible for the polyclonal B-cell activation observed. The aim of this work was to test the effects of aurothiopropanolsulphonate (ATPS) in BN and LEW rats. In BN rats, ATPS induced a polyclonal B-cell activation marked by lymphoproliferation, hyperimmunoglobulinaemia affecting mainly IgE, and by the production of numerous autoantibodies. A glomerulonephritis occurred, initially due to anti-glomerular basement membrane antibody deposition, and later to the formation of granular deposits, occasionally resulting in a typical membranous glomerulopathy. Self class-II-specific T-cells were found that might be responsible for the polyclonal B-cell activation. Lewis rats were free of glomerulopathy but, like BN rats, exhibited an interstitial nephritis and some degree of polyclonal B-cell activation. These findings demonstrate that, depending on the strain, ATPS triggers different B-cell clones inducing different degrees of autoimmunity.

Evaluation of the developmental toxicity of 2,3-dimercapto-1-propanesulfonate (DMPS) in mice. Effect on mineral metabolism.

The sodium salt of 2,3-dimercapto-1-propanesulfonic acid (DMPS), a potent chelating agent used in the treatment of inorganic and organic heavy metal intoxications was evaluated for developmental toxicity in pregnant Swiss mice. DMPS was administered by gavage at doses of 0, 75, 150 and 300 mg/kg per day on gestational days 6-15. Females were evaluated for body weight gain, food consumption, appearance and behavior, survival rates and reproduction data. Cesarean sections were performed on gestation day 18. There were no maternal toxic effects, and no treatment-related changes were recorded in the number of total implants, resorption, the number of live and dead fetuses, fetal body weight or fetal sex distribution data. Gross external, soft tissue and skeletal examination of the DMPS-treated fetuses did not show significant differences at any dose in comparison with the controls. Mineral analysis of maternal and fetal tissues revealed slight effects of DMPS on metabolism of calcium, magnesium, zinc, copper and iron. The results of this study in mice indicate that DMPS is not a developmental toxicant at levels up to 300 mg/kg per day.

2,3-Dithioerythritol, a possible new arsenic antidote.

British antilewisite (2,3-dimercaptopropanol; BAL) has long been used as an arsenic antidote, but its therapeutic efficacy is limited by its inherent toxicity. We synthesized two less toxic derivatives of BAL and investigated their potential as antidotes to organic arsenic. The new compounds, 2,3-dithioerythritol (DTE) and 2,2-dimethyl-4-(hydroxymethyl)-1,3-dithiolane (isopropylidene derivative of BAL), react readily with phenyldichloroarsine (PDA) to yield the expected corresponding cyclic 1,3-dithioarsolanes. The BAL derivatives were compared to BAL in terms of their cytotoxicity and their ability to rescue PDA-poisoned mouse lymphoma cells in culture. The dithiolane was not a good antidote in the cultured cell system. In contrast, DTE was less toxic than BAL or DMSA and was superior at improving cell survival in PDA-exposed cells.

N-(2,3-dimercaptopropyl)phthalamidic acid: protection, in vivo and in vitro, against arsenic intoxication.

The ip LD50s of N-(2,3-dimercaptopropyl)phthalamidic acid (DMPA) and British Anti-Lewisite (BAL) were 0.819 and 1.48 mmol/kg, respectively, in male albino mice. The ip ED50 of DMPA and BAL for prevention of the lethal effects of 0.15 mmol NaAsO2/kg was 0.022 and 0.169 mmol/kg, respectively. DMPA increased the LD50 of sodium arsenite by approximately 2.5-fold following two ip injections of 0.20 mmol DMPA/kg. The effectiveness of DMPA in reducing the toxicity of NaAsO2 was further demonstrated by its reversal of the sodium arsenite inhibition of pyruvate dehydrogenase multienzyme complex (PDH) activity in vitro. Similarly, in an in vivo experiment in which mice received 0.10 mmol NaAsO2/kg, and 30 min later were given 0.05 or 0.10 mmol/kg DMPA, there was a rapid recovery of PDH activity. The distribution of 74As in the tissues of male New Zealand rabbits was altered following im injection of 0.20 mmol/kg DMPA. Under these conditions, the tissue concentration of 74As was significantly decreased. For all tissues tested, the 74As content decreased by at least 50% as compared to that of untreated controls. DMPA was effective also in increasing both urinary and fecal excretion of arsenic. The stability of aqueous solutions of DMPA varies with the pH of the solution. DMPA is more stable in acid solution.

A methylmercury toxicity model to test for possible adverse effects resulting from chelating agent therapy.

A daily dosing model for methylmercury (MM) intoxication was developed for the purpose of testing for possible adverse effects resulting from the administration of complexing agents used in the treatment of MM poisoning. The dithiol complexing agents 2,3-dimercaptopropanol (BAL) and meso-2,3-dimercaptosuccinic acid (DMSA) were chosen to test the discriminative ability of this model, since the former is contraindicated for MM poisoning and causes an increase in target organ MM burden, while the latter compound is known to be efficacious in reducing both toxicity and brain MM content. The basic design of the model called for daily observation of treated animals with identification of the following signs of MM intoxication: loss of body weight, onset of signs of toxicity, and mortality. The degree of toxicity was evaluated, and a toxicity score (0-5) was provided for each animal. A dose-dependent decrease in body weight was found in MM-treated mice. The latent period for development of signs of intoxication varied inversely with the dose rate. The rate of progression of severity of signs of intoxication was also dependent upon the dose. A dose rate of 14 mg Hg per kg per day was utilized to test the effects of BAL and DMSA on the onset and progression of signs of MM intoxication. Onset and progression of signs of methylmercury intoxication were similar for animals receiving methylmercury either alone or with administration of BAL at 2 mg per kg per day. Animals which received BAL at a dose rate of 20 mg per kg per day developed signs of intoxication significantly earlier.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the oxygen toxicity after administration of chelate-forming agents in mice.

The influence of the chelating agents CaNa2-EDTA, penicillamine, dimercaprol, unithiol, dithiocarb, aurin tricarboxylic acid, salicylic acid, and acetylsalicylic acid on the toxicity of oxygen at elevated tension was studied in mice. With the possible exception of dimercaprol, no increase in toxic effects of oxygen was observed after administering these chelate-forming agents in the doses used.

Toxicological studies on sodium 2,3-dimercaptopropane-1-sulfonate in the rat.

The toxicity of Na-2,3-dimercaptopropane-1-sulfonate (DMPS) was investigated in rats. The lethality after large i.p. doses is diminished by a factor of approx. 6 if a single dose is divided into several doses on consecutive days. Chronic treatment with 600 mumol DMPS/kg and day p.o. did bring about adverse effects except a reduction in Cu-concentration in some organs, especially in the kidneys, immediately after DMPS-treatment, which was normalized within 1 week. The offspring of DMPS treated female rats showed no abnormalities.