The processes and mechanism of antimony sequestered by red blood cells and its metabolic conjugation with hemoglobin in rats.

Toxicity of antimony (Sb) to humans may occur through environmental, occupational, and therapeutic exposures. The underlying mechanism of its accumulation in red blood cells (RBCs) is not clear. Here, the processes and mechanism of RBCs sequestering Sb were explored through a series of in vitro and in vivo studies. These include binding affinity of Sb compounds with rat hemoglobin (Hb) and RBCs, acute exposure of antimony potassium tartrate (APTIII), and subchronic oral exposure of APTIII, potassium pyroantimonate (PPV) and antimony trioxide (ATIII). In vitro study indicated that parent form of Sb exhibited notable affinity with RBCs, while represented negligible affinity with Hb. However, after acute exposure to APTIII, over 93% of Sb in rat blood was integrated into Hb. Sb retaining in liver homogenate supernatants was substantially integrated into Hb, which indicated liver metabolism played a potential role influencing its ultimate partitioning in blood. Subchronic exposure of APTIII, PPV and ATIII to rats also demonstrated that most of Sb metabolites were integrated into Hb, regardless of which Sb compounds administered. Nano-HPLC-MS/MS analysis suggested that a dimethylated Sb species in pentavalent state [Sb(CH3)2O2H] was a major Sb group conjugated with Hb at Cys 104 and Cys 111 especially, in α chain of rat Hb. Hb-conjugated Sb did not affect Hb's oxygen binding capability. As the deposit sites for both the parent forms and the metabolite, Sb re-released to circulation system due to RBCs and Hb degradation could have high substantial toxicological effects on its potential sites of action.

Generation of an aquaglyceroporin AQP1 null mutant in Leishmania major.

The Leishmania aquaglyceroporin AQP1 plays an important physiological role in water and uncharged polar solutes transport, volume regulation, osmotaxis, and is a key determinant of antimony resistance. By targeted gene disruption, we generated a Leishmania major promastigote AQP1 null mutant. This required several attempts but a chromosomal null AQP1 mutant was obtained by loss of heterozygosity in the presence of a rescue plasmid encoding AQP1. Growth in the absence of selection led to the loss of the rescuing plasmid, indicating that AQP1 is not essential for Leishmania viability. The AQP1-null mutant was resistant to antimonyl tartrate (SbIII) and arsenite (AsIII) due to a decrease import of these metalloids. It also exhibited alterations in its osmoregulation abilities compared with wild-type cells. This is the first report of the generation of a genetic AQP1 null mutant in Leishmania parasite, confirming its physiological function and role in resistance to antimonials, the therapeutic mainstay against Leishmania.

Reduced cardiovascular alterations of tartar emetic administered in long-circulating liposomes in rats.

Trivalent antimonial drugs, including tartar emetic (TA), are known to induce important cardiotoxicity observed by electrocardiographic abnormalities. Liposome encapsulation was found to reduce the overall acute toxicity of TA. The present work investigated the cardiovascular parameters alterations of rats submitted to the treatment with free and encapsulated TA in long-circulating liposomes. Liposomes were made using lipids DSPC, DSPE-PEG and cholesterol. The cardiovascular signals, electrocardiogram (ECG) and arterial blood pressure (AP), were recorded from anaesthetized Wistar rats after intravenous (IV) administration of a single specially high dose (17 mg/kg) of TA in liposomes and in free form. The IV administration of TA solution caused significant increase of QT interval of ECG and significant reduction of AP when compared to the control group. These alterations were not observed when liposomes TA were administered and the profile of ECG and AP data was quite similar to the control groups. In conclusion, a liposomal formulation of TA showed a reduced cardiotoxic profile for TA when compared to the free form.

Effects of antimony on aquatic organisms (Larva and embryo of Oryzias latipes, Moina macrocopa, Simocephalus mixtus, and Pseudokirchneriella subcapitata).

Antimony is widespread in aquatic environment. Trivalent forms of antimony are known to be more toxic than other chemical species of antimony. In the present study, antimony potassium tartrate (APT), the trivalent inorganic forms of antimony, was selected as a test antimony compound due to its high water solubility. The effects of antimony on Japanese medaka (Oryzias latipes), planktonic crustacea (Moina macrocopa and Simocephalus mixtus), and green algae (Pseudokirchneriella subcapitata) were evaluated. Larval survival and the embryonic development were measured for fish assay. APT was less toxic to larval medaka (24-h LC50, 261; 48-h LC50, 238 mg L(-1)). Simocephalus mixtus was killed by very low concentrations of APT (24-h LC50, 4.92 mg L(-1)), and antimony was also toxic to Moina macrocopa (24-h LC50, 12.83 mg L(-1)). Toxicities of APT to S. mixtus and Moina macrocopa were about 50 and 20 times more toxic to Oryzias latipes larvae, respectively, in terms of 24-h LC50 value. Growth inhibition of Pseudokirchneriella subcapitata was observed in the presence of APT (72-h EC50, 206 mg L(-1)). This study demonstrated that APT is more toxic to planktonic crustacea than fish and green algae, and planktonic crustacea appears a better indicator of antimony pollution in aquatic environment.

Antimony toxicity from the use of tartar emetic for the treatment of alcohol abuse.

Antimony is a poisonous element with toxic properties that mimic those of arsenic. Numerous reports describe gastrointestinal complications of vomiting, diarrhea and stomatitis associated with antimony exposure. However, antimony toxicity from the use of tartar emetic as a treatment for alcohol abuse has never been described previously. A 19-y-o man with a history of alcohol abuse ingested a 10 mL bottle of "Soluto Vital" (tartar emetic, 50 mg/mL), produced in Guatemala for treatment of alcohol abuse. He presented 60 min after ingestion with severe vomiting, abdominal cramps, diarrhea, weakness and orthostasis. Initial laboratory evaluations were remarkable for creatinine of 2.5 mg/dL, potassium 6.1 mEq/L, and 60% hematocrit. He was given activated charcoal, iv saline and antiemetics. Over the next 48 h his creatinine normalized to 1.1 mg/dL and the hematocrit returned to 53%; urine had an antimony concentration of 1200 mcg/L (normal = < 10 mcg/L). It is important to recognize that foreign alcohol therapies aversive therapy other than disulfiram may be used, the contents of such a foreign product should be identified.

Role of the Multidrug Resistance Protein 1 in protection from heavy metal oxyanions: investigations in vitro and in MRP1-deficient mice.

The Multidrug Resistance Protein 1 (MRP1) is a membrane pump that mediates the efflux of a wide variety of xenobiotics, including arsenical and antimonial compounds, as demonstrated by the study of MRP1-transfected cell lines. We have previously shown that mrp1(-/-) cells are hypersensitive to sodium arsenite, sodium arsenate, and antimony potassium tartrate. We now report that the retroviral vector-mediated overexpression of MRP1 and of the two subunits of gamma-GCS (heavy and light) resulted in higher intracellular glutathione levels and in a greater level of resistance to sodium arsenite and antimony potassium tartrate, compared to the overexpression of MRP1 and gamma-GCS heavy alone. These observations further demonstrate that glutathione is an important component of MRP1-mediated cellular resistance to arsenite and antimony. However, the constitutive expression of MRP1 did not protect mice from the lethality of sodium arsenite and antimony potassium tartrate nor reduced the tissue accumulation of arsenic in mice injected i.p. with sodium arsenite. It is conceivable that, in vivo, other pump(s) effectively vicariate for MRP1-mediated transport of heavy metal oxyanions.

Induction of stress proteins in rat cardiac myocytes by antimony.

The effects of nonlethal concentrations of potassium antimonyl tartrate (PAT) were examined in cultured neonatal rat cardiac myocytes. PAT (5, 10 microM) significantly increased cellular reduced glutathione (GSH) and heme oxygenase activity after 18 h. GSH levels and heme oxygenase activity were increased 2.5- and 5.4-fold, respectively, by 10 microM PAT after 18 h. In addition, total cytochrome P450 levels were decreased by PAT after an 18-h exposure. PAT exposures were associated with the induction of specific stress proteins. Nonlethal concentrations of PAT produced a dose-dependent increase in HO-1, HSP70, and HSP25/27 protein levels but did not increase HSP60 levels. Pretreatment of cardiac myocytes with low concentrations of PAT (0.5-10 microM) protected against a subsequent lethal concentration of PAT (200 microM). This protection was blocked if cells were treated with the protein synthesis inhibitor cycloheximide. Results demonstrate that low concentrations of PAT increase GSH levels and stress protein synthesis, which may be responsible for the protection that low-level PAT exposure offers against the subsequent toxicity of higher concentrations of PAT.

Review of subchronic/chronic toxicity of antimony potassium tartrate.

Subchronic/chronic toxicity studies on antimony potassium tartrate (APT) have been reviewed. One of the older studies (H. A. Schroeder et al., 1970, J. Nutr. 100 (1), 59-68), on which are based the EPA reference dose value and a number of state, national, and international drinking water criteria for antimony, has severe inadequacies in study conduct making it uninterpretable and inappropriate for characterization of APT toxicity. In particular, the manner in which control data were generated and utilized in this study is considered invalid. More recent drinking water studies conducted by the NTP (1992, "NTP Technical Report on Toxicity Studies of Antimony Potassium Tartrate in F344/N Rats and B6C3F(1) Mice (Drinking Water and Intraperitoneal Injection Studies)," NTP Toxicity Report Series, No. 11) and Poon et al. (1998, Food Chem. Toxicol. 36, 20-35) showed antimony to be of low toxicity. The NOAEL in the 14-day NTP study was 2500 ppm by the oral route in both rats and mice, while Poon et al. (1998) suggested a NOAEL of 0.5 ppm in their 90-day study. However, upon close examination, it was determined that this value was based on subtle histological changes in the thyroid gland that were physiological, not toxicological, in nature. This conclusion is supported further by an absence of these changes in a well-conducted 13-week intraperitoneal exposure study in rats that utilized APT at much higher doses (NTP, 1992). Thus, the NOAEL by Poon et al. (1998) should more appropriately be 50 ppm. When regulatory criteria for antimony are established and/or reviewed, the findings in the NTP study and this critical reevaluation of the Poon et al. (1998) study should be considered when establishing a NOAEL for subchronic exposure to antimony in the future.

The role of intracellular calcium in antimony-induced toxicity in cultured cardiac myocytes.

Trivalent antimony, delivered as potassium antimonyl tartrate (PAT), has been previously shown to induce an oxidative stress and toxicity in cultured neonatal rat cardiac myocytes. The present study investigates the effect of PAT on intracellular free calcium ([Ca2+]i), which has been implicated in the toxicity of agents inducing oxidative stress, and explores its role in PAT toxicity. Exposure to 50 or 200 microM PAT led to progressive elevation in diastolic or resting [Ca2+]i and eventually a complete loss of [Ca2+]i transients that occurred well before cell death as assessed by LDH release. Prior loading of myocytes with the intracellular calcium chelator BAPTA (10 to 40 microM), protected against PAT toxicity in the presence and absence of extracellular calcium, and demonstrated a crucial role for [Ca2+]i in PAT toxicity. Exposure to 200 microM PAT in the absence of extracellular calcium slightly elevated [Ca2+]i, but only to levels comparable to resting [Ca2+]i for cells in 1.8 mM extracellular calcium. This demonstrated that although PAT toxicity was dependent on [Ca2+]i, a large increase above resting levels was not needed, and also that some calcium was mobilized from intracellular stores. However, the caffeine-releasable pool of sarcoplasmic reticulum calcium was increased, not depleted, by exposure to 200 microM PAT. These results demonstrate that PAT disrupts [Ca2+]i handling and support a role for a calcium-dependent event, but do not support the necessity of events in PAT-induced cell death that are mediated by a large elevation in [Ca2+]i.

Antimony-induced alterations in thiol homeostasis and adenine nucleotide status in cultured cardiac myocytes.

Cultured cardiac myocytes were exposed for up to 4 h to 50 and 100 microM potassium antimonyl tartrate (PAT). After 4 h, 50 and 100 microM PAT killed 14 and 33% respectively of the cardiac myocytes. PAT-induced alterations in both protein and nonprotein thiol homeostasis. Transient increases in oxidized glutathione disulfide (GSSG) levels were detected after cells were treated with 100 microM PAT for 2 h. After 4 h, both concentrations of PAT significantly depleted reduced glutathione (GSH) levels. Protein thiols levels were also decreased after a 2-h exposure to 50 and 100 microM PAT. Cells treated with 50 microM and 100 microM PAT had a 15% and 40% reduction respectively in protein thiols after 4 h. PAT also significantly inhibited glutathione peroxidase and pyruvate dehydrogenase activity in cardiac myocytes. Pyruvate dehydrogenase activity levels were inhibited as early as 1 h after cells were treated with both concentrations of PAT. Cardiac myocyte ATP levels were also decreased by PAT, but only after a 4-h exposure to 50 microM and 100 microM PAT. Decreases in cellular ATP levels paralleled PAT toxicity put appeared to be secondary to other cellular changes initiated by PAT exposure.

Cross-resistance between cisplatin, antimony potassium tartrate, and arsenite in human tumor cells.

Cross-resistance between cisplatin (DDP) and metalloid salts in human cells was sought on the basis that mechanisms that mediate metalloid salt cross-resistance in prokaryotes are evolutionarily conserved. Two ovarian and two head and neck carcinoma cell lines selected for DDP resistance were found to be cross-resistant to antimony potassium tartrate, which contains trivalent antimony. The DDP-resistant variant 2008/A was also cross-resistant to arsenite but not to stibogluconate, which contains pentavalent antimony. A variant selected for resistance to antimony potassium tartrate was cross-resistant to DDP and arsenite. Resistance to antimony potassium tartrate and arsenite was of a similar magnitude (3-7-fold), whereas the level of resistance to DDP was greater (17-fold), irrespective of whether the cells were selected by exposure to DDP or to antimony potassium tartrate. In the resistant sublines, uptake of [3H]-dichloro(ethylenediamine) platinum(II) was reduced to 41-52% of control, and a similar deficit was observed in the accumulation of arsenite. We conclude that DDP, antimony potassium tartrate, and arsenite all share a common mechanism of resistance in human cells and that this is due in part to an accumulation defect.

Antimony-induced oxidative stress and toxicity in cultured cardiac myocytes.

Cardiac myocytes were exposed to concentrations of potassium antimonyl tartrate (PAT) ranging from 1 to 1000 microM for 1 to 24 hr. Toxicity was assessed by measuring lactate dehydrogenase (LDH) release and by monitoring chronotropic depression. Lipid peroxidation was assessed by measuring the release of thiobarbituric acid reactive substances (TBARS). PAT produced a concentration- and time-dependent depression in chronotropy and an increase in the release of LDH and TBARS. A 4-hr exposure to 100 microM PAT stopped beating and induced significant increases in TBARS and LDH release in the myocyte cultures. The lipid peroxidation and LDH release induced by 100-200 microM PAT at 4 hr could be prevented by pretreatment of the cardiac myocytes with vitamin E or by the simultaneous addition of other antioxidants. Vitamin E continued to protect against lipid peroxidation up to 18 hr after the addition of 100 microM PAT, but failed to provide significant protection against LDH release at this time-point. Both 50 and 100 microM PAT decreased cardiac myocyte glutathione (GSH) levels after a 4-hr exposure. A series of thiol-containing compounds was evaluated for their effects on PAT toxicity. The addition of dithiothreitol, GSH, and 2-mercaptoethanol afforded some degree of protection against lipid peroxidation and LDH release up to 18 hr after the addition of 100 microM PAT. These results suggest that PAT induces lipid peroxidation in cultured cardiac myocytes but that other mechanisms may contribute to cell death with long-term exposures to PAT. Our results also suggest that PAT interacts with thiol-containing compounds.

Comparative toxicity and tissue distribution of antimony potassium tartrate in rats and mice dosed by drinking water or intraperitoneal injection.

Antimony potassium tartrate (APT) is a complex salt that until recently was used worldwide as an antischistosomal drug. Treatment was efficacious only if APT was administered intravenously to humans at a near lethal total dose of 36 mg/kg. Because unconfirmed epidemiologic studies suggested there might be an association between APT treatment and bladder cancer, we initiated prechronic toxicity studies with the drug to select a route of administration and doses in the event that chronic studies of APT were needed. The toxicity and concentration of tissue antimony levels were compared in 14-d studies with F344 rats and B6C3F1 mice administered APT in the drinking water or by ip injection to determine the most appropriate route for longer term studies. Drinking water doses estimated by water consumption were 0, 16, 28, 59, 94 and 168 mg/kg in rats and 0, 59, 98, 174, 273, and 407 mg/kg in mice. APT was poorly absorbed and relatively nontoxic orally, whereas ip administration of the drug caused mortality, body weight decrements, and lesions in the liver and kidney at doses about one order of magnitude below those in drinking water. Because of these data and the dose-related accumulation of antimony in the target organs, an ip dose regimen was selected for subsequent studies. Both sexes of F344 rats and B6C3F1 mice were given 0, 1.5, 3, 6, 12, and 24 mg/kg doses of APT every other day for 90 d by ip injection. There were no clinical signs of toxicity nor gross or microscopic lesions in mice that could be attributed to toxicity of APT, although elevated concentrations of antimony were detected in the liver and spleen of mice. Rats were more sensitive than mice to the toxic effects of APT, exhibiting dose-related mortality, body weight decrements, and hepatotoxicity. The concentrations of antimony measured in liver, blood, kidney, spleen, and heart of rats were proportional to dose, but there were no biochemical changes indicative of toxicity except in the liver. Hepatocellular degeneration and necrosis occurred in association with dose-related elevations in activities of the liver-specific serum enzymes sorbitol dehydrogenase and alanine aminotransferase. By alternating the site of abdominal injection and the days of treatment, mesenteric inflammation at the site of administration was minimized in the rats and mice, indicating that the ip route would be suitable for chronic studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytogenetic effect of two antimonial antibilharzial drugs: tartar emetic and bilharcid.

The effects of tartar emetic and bilharcid, two antimonial antibilharzial drugs, on the chromosomes of laboratory rats are studied. The drugs were administered intraperitoneally in three doses--clinical, intermediate, and maximum tolerated--both acutely (6, 24, and 48 hours) and subacutely. The two drugs produced the same types of chromosomal aberrations with tartar emetic, inducing a higher rate of incidence. No significant differences in the number of cells with chromosomal aberrations were generally observed among the 6, 24, and 48 hours of the acute treatment with both tartar emetic and bilharcid. The dose-response relationship was examined for both the acute and the subacute treatments. Whereas the acute treatment of tartar emetic showed a dose-dependent linear increase in the number of cells with chromosomal aberrations, the subacute treatment of tartar emetic and the acute and subacute treatments of bilharcid displayed their maximum effects at an intermediate dose.

Effect of cysteine on the hepatic toxicity and antischistosomal activity of antimonyl potassium tartrate.

Cysteine produced a significant and progressive reduction in the toxicity of antimonyl potassium tartrate (APT) when the two substances were injected into mice and rabbits in ratios of APT to cysteine ranging from 1:1 to 1:3. The reduction in toxicity was highest with the 1:3 ratio. However, the combination of the two substances, especially in the ratio of 1:3, appreciably reduced the antischistosomal activity of APT both in vivo and in vitro.