Cytotoxic Effect of Lipophilic Bismuth Dimercaptopropanol Nanoparticles on Epithelial Cells.

Bismuth nanoparticles have many interesting properties to be applied in biomedical and medicinal sectors, however their safety in humans have not been comprehensively investigated. The objective of this research was to determine the cytotoxic effect of bismuth dimercaptopropanol nanoparticles (BisBAL NPs) on epithelial cells. The nanoparticles are composed of 18.7 nm crystallites on average and have a rhombohedral structure, agglomerating into chains-like or clusters of small nanoparticles. Based on MTT viability assay and fluorescence microscopy, cytotoxicity was not observed on monkey kidney cells after growing with 5 µM of BisBAL NPs for 24 h. Employing same techniques, identical results were obtained with human epithelial cells (HeLa), showing a not strain-dependent phenomenon. The absence of toxic effects on epithelial cells growing with BisBAL NPs was corroborated with long-time experiments (24-72 hrs.), showing no difference in comparison with growing control (cells without nanoparticles). Further, genotoxicity assays, comet assay and fluorescent microscopy and electrophoresis in bromide-stained agarose gel revealed no damage to genomic DNA of MA104 cells after 24 h. of exposition to BisBAL NPs. Finally, the effect of bismuth nanoparticles on protein synthesis was studied in cells growing with BisBAL NPs for 24 h. SDS-PAGE assays showed no difference between treated and untreated cells, suggesting that BisBAL NPs did not interfere with protein synthesis. Hence BisBAL NPs do not appear to exert cytotoxic effects suggesting their biological compatibility with epithelial cells.

Antimicrobial and antitumor activities of an alginate-based membrane loaded with bismuth nanoparticles and cetylpyridinium chloride.

OBJECTIVE: To evaluate the antitumor and antimicrobial properties of an alginate-based membrane (ABM) loaded with bismuth lipophilic nanoparticles (BisBAL NPs) and cetylpyridinium chloride (CPC) on clinically isolated bacteria and a pancreatic cancer cell line. MATERIAL AND METHODS: The BisBAL NP-CPC ABM was characterized using optical and scanning electron microscopy (SEM). The antimicrobial potential was measured using the disk-diffusion assay, and antibiofilm activity was determined through the live/dead assay and fluorescence microscopy. The antitumor activity was analyzed on the pancreatic cell line (Panc 03.27) using the MTT assay and live/dead assay with fluorescence microscopy. RESULTS: After a 24-h exposure (37°C, aerobic conditions), 5 µM BisBAL NP reduced the growth of K. pneumoniae by 77.9%, while 2.5 µM BisBAL NP inhibited the growth of Salmonella, E. faecalis and E. faecium by 82.9%, 82.6%, and 78%, respectively (p < 0.0001). The BisBAL NPs-CPC ABM (at a ratio of 10:1; 500 and 50 µM, respectively) inhibited the growth of all isolated bacteria, producing inhibition halos of 9.5, 11.2, 7, and 10.3 mm for K. pneumoniae, Salmonella, E. faecalis, and E. faecium, respectively, in contrast to the 6.5, 9.5, 8.5, and 9.8 mm obtained with 100 µM ceftriaxone (p < 0.0001). The BisBAL NPs-CPC ABM also reduced bacterial biofilms, with 81.4%, 74.5%, 97.1%, and 79.5% inhibition for K. pneumoniae, E. faecium, E. faecalis, and Salmonella, respectively. Furthermore, the BisBAL NPs-CPC ABM decreased Panc 03.27 cell growth by 76%, compared to 18% for drug-free ABM. GEM-ABM reduced tumoral growth by 73%. The live/dead assay confirmed that BisBAL NPs-CPC-ABM and GEM-ABM were cytotoxic for the turmoral Panc 03.27 cells. CONCLUSION: An alginate-based membrane loaded with BisBAL NP and CPC exhibits dual antimicrobial and antitumoral efficacy. Therefore, it could be applied in cancer treatment and to diminish the occurrence of surgical site infections.

Bismuth Lipophilic Nanoparticles (BisBAL NP) Inhibit the Growth of Tumor Cells in a Mouse Melanoma Model.

AIM: The objective of this study was to analyze the antitumor effect of BisBAL NP in a mouse melanoma model. MATERIALS AND METHODS: The antitumor activity of BisBAL NP on murine B16-F10 melanoma cells was determined both in vitro (PrestoBlue cell viability assay and Live/Dead fluorescence) and in vivo, in a mouse model, with the following 15-day treatments: BisBAL NP, negative control (PBS), and cell-death control (docetaxel; DTX). Mouse survival and weight, as well as the tumor volume, were recorded daily during the in vivo study. RESULTS: BisBAL NP were homogeneous in size (mean diameter, 14.7 nm) and bismuth content. In vitro, 0.1 mg/mL BisBAL NP inhibited B16-F10 cell growth stronger (88%) than 0.1 mg/mL DTX (82%) (*p<0.0001). In vivo, tumors in mice treated with BisBAL NP (50 mg/kg/day) or DTX (10 mg/kg/day) were 76% and 85% smaller than the tumors of negative control mice (*p<0.0001). The average weight of mice was 18.1 g and no statistically significant difference was detected among groups during the study. Alopecia was only observed in all DTX-treated mice. The survival rate was 100% for the control and BisBAL NP groups, but one DTX- treated mouse died at the end of the treatment period. The histopathological analysis revealed that exposure to BisBAL NP was cytotoxic for tumor tissue only, without affecting the liver or kidney. CONCLUSION: BisBAL NP decreased the tumor growing in a mouse melanoma model without secondary effects, constituting an innovative low-cost alternative to treat melanoma.

In vitro efficacy of bismuth thiols against biofilms formed by bacteria isolated from human chronic wounds.

AIMS: The purpose of this study was to evaluate the antimicrobial efficacy of thirteen bismuth thiol preparations for bactericidal activity against established biofilms formed by two bacteria isolated from human chronic wounds. METHODS: Single species biofilms of a Pseudomonas aeruginosa or a methicillin-resistant Staphylococcus aureus were grown in either colony biofilm or drip-flow reactors systems. Biofilms were challenged with bismuth thiols, antibiotics or silver sulfadiazine, and log reductions were determined by plating for colony formation. CONCLUSIONS: Antibiotics were ineffective or inconsistent against biofilms of both bacterial species tested. None of the antibiotics tested were able to achieve >2 log reductions in both biofilm models. The 13 different bismuth thiols tested in this investigation achieved widely varying degrees of killing, even against the same micro-organism in the same biofilm model. For each micro-organism, the best bismuth thiol easily outperformed the best conventional antibiotic. Against P. aeruginosa biofilms, bismuth-2,3-dimercaptopropanol (BisBAL) at 40-80 µg ml⁻¹ achieved > 7·7 mean log reduction for the two biofilm models. Against MRSA biofilms, bismuth-1,3-propanedithiol/bismuth-2-mercaptopyridine N-oxide (BisBDT/PYR) achieved a 4·9 log reduction. SIGNIFICANCE AND IMPACT OF THE STUDY: Bismuth thiols are effective antimicrobial agents against biofilms formed by wound bacteria and merit further development as topical antiseptics for the suppression of biofilms in chronic wounds.

Bismuth dimercaptopropanol (BisBAL) inhibits formation of multispecies wastewater flocs.

AIMS: To determine the ability of a bismuth thiol to control floc formation in a multispecies population of micro-organisms obtained from the activated sludge unit of a wastewater treatment plant. The molecular level mechanisms by which bismuth-2-3-dimercapto-1-propanol (BisBAL) inhibits bioaggregation are also elucidated. METHODS AND RESULTS: Micro-organisms were grown over a 3-day period in a batch system by adding glucose as an electron donor to stimulate short-term heterotrophic activity. Extracellular polymeric substances (EPS) produced by activated sludge micro-organisms during exponential and stationary growth phases in the presence and absence of BisBAL were characterized using colorimetry, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FTIR) spectroscopy. BisBAL at its minimum inhibitory concentration (MIC, 10 µmol l(-1) ) was most effective in suppressing microbial floc formation. The principal effect of sub-inhibitory concentrations of BisBAL was to decrease total EPS production while largely preserving homology. CONCLUSIONS: Antifouling and bactericidal properties of BisBAL arise from its ability to reduce EPS expression and preferentially suppressing acidic and O-acetylated carbohydrates and certain protein secondary structures viz. ß-structures, random coils, and α-and 3-turn helices. As micro-organisms exhibited a much weaker tendency to aggregate at lower concentrations of these specific EPS components, they also appear to be important for the formation of microbial flocs and bioaggregates. SIGNIFICANCE AND IMPACT OF THE STUDY: BisBAL was shown to be highly effective against multispecies microbial aggregation. Novel bismuth-based biocides could also be potentially employed to control excess sludge production in wastewater treatment systems by inhibiting EPS expression.

Arsenic Toxicity: Molecular Targets and Therapeutic Agents.

: High arsenic (As) levels in food and drinking water, or under some occupational conditions, can precipitate chronic toxicity and in some cases cancer. Millions of people are exposed to unacceptable amounts of As through drinking water and food. Highly exposed individuals may develop acute, subacute, or chronic signs of poisoning, characterized by skin lesions, cardiovascular symptoms, and in some cases, multi-organ failure. Inorganic arsenite(III) and organic arsenicals with the general formula R-As2+ are bound tightly to thiol groups, particularly to vicinal dithiols such as dihydrolipoic acid (DHLA), which together with some seleno-enzymes constitute vulnerable targets for the toxic action of As. In addition, R-As2+-compounds have even higher affinity to selenol groups, e.g., in thioredoxin reductase that also possesses a thiol group vicinal to the selenol. Inhibition of this and other ROS scavenging seleno-enzymes explain the oxidative stress associated with arsenic poisoning. The development of chelating agents, such as the dithiols BAL (dimercaptopropanol), DMPS (dimercapto-propanesulfonate) and DMSA (dimercaptosuccinic acid), took advantage of the fact that As had high affinity towards vicinal dithiols. Primary prevention by reducing exposure of the millions of people exposed to unacceptable As levels should be the prioritized strategy. However, in acute and subacute and even some cases with chronic As poisonings chelation treatment with therapeutic dithiols, in particular DMPS appears promising as regards alleviation of symptoms. In acute cases, initial treatment with BAL combined with DMPS should be considered.

Long-term MBR performance of polymeric membrane modified with Bismuth-BAL chelate (BisBAL).

An ultrafiltration membrane prepared by polyethersulfone (PES) was modified with Bismuth-BAL chelate (BisBAL) and was used in submerged membrane bioreactor system. Moreover, a control membrane reactor was also tasked to evaluate the effect of BisBAL on the membrane performance. The flux profile, transmembrane pressure, the effect of chemical treatment, cake layer formation, anti-fouling properties against extracellular polymeric substances (EPS) and soluble microbial products (SMP) were studied. The UF modified membrane demonstrated a sustained permeability, low cleaning frequency, and longer filtration time. In terms of anti-EPS and SMP accumulation, the modified membrane showed a lower membrane resistance. It can be illustrated from scanning electron microscopy and confocal laser scanning microscope images that the modified membrane had presented better properties than bare PES membrane, as it was looser and thinner. Thus, the UF membrane proved to be more efficient in terms of permeability and lifetime.

Improvement of anti-biofouling properties of hollow fiber membranes with bismuth-BAL chelates (BisBAL).

In this study, hollow fiber membranes with and without bismuth dimercaptopropanol (Bis-BAL) additive were fabricated. Membranes were characterized in terms of permeability, surface properties, anti-biofouling and antibacterial properties. Membranes were operated in a lab-scale submerged membrane bioreactor (MBR). During the MBR operation, flux, chemical oxygen demand, volatile suspended solids and suspended solids were calculated for 30 days. Results showed that extracellular polymeric substance and soluble microbial product amounts were decreased in BisBAL-containing membranes. BisBAL-added membranes had the ability to inhibit the growth of Escherichia coli. BisBAL as an additive for membranes was found to be an effective, cheap alternative for enhancing anti-biofouling properties of the membranes.

Bismuth dimercaptopropanol (BisBAL) inhibits the expression of extracellular polysaccharides and proteins by Brevundimonas diminuta: implications for membrane microfiltration.

A 2:1 molar ratio preparation of bismuth with a lipophilic dithiol (3-dimercapto-1-propanol, BAL) significantly reduced extracellular polymeric substances (EPS) expression by Brevundimonas diminuta in suspended cultures at levels just below the minimum inhibitory concentration (MIC). Total polysaccharides and proteins secreted by B. diminuta decreased by approximately 95% over a 5-day period when exposed to the bismuth-BAL chelate (BisBAL) at near MIC (12 microM). Fourier-transform infrared spectroscopy (FTIR) suggested that a possible mechanism of biofilm disruption by BisBAL is the inhibition of carbohydrate O-acetylation. FTIR also revealed extensive homology between EPS samples with and without BisBAL treatment, with proteins, polysaccharides, and peptides varying predominantly only in the amount expressed. EPS secretion decreased following BisBAL treatment as verified by atomic force microscopy and scanning electron microscopy. Without BisBAL treatment, a slime-like EPS matrix secreted by B. diminuta resulted in biofouling and inefficient hydrodynamic backwashing of microfiltration membranes.

In vitro evaluation of the antitumor effect of bismuth lipophilic nanoparticles (BisBAL NPs) on breast cancer cells.

AIM: The objective of this study was to evaluate the antitumor activity of lipophilic bismuth nanoparticles (BisBAL NPs) on breast cancer cells. MATERIALS AND METHODS: The effect of varying concentrations of BisBAL NPs was evaluated on human MCF-7 breast cancer cells and on MCF-10A fibrocystic mammary epitheliocytes as noncancer control cells. Cell viability was evaluated with the MTT assay, plasma membrane integrity was analyzed with the calcein AM assay, genotoxicity with the comet assay, and apoptosis with the Annexin V/7-AAD assay. RESULTS: BisBAL NPs were spherical in shape (average diameter, 28 nm) and agglomerated into dense electronic clusters. BisBAL NP induced a dose-dependent growth inhibition. Most importantly, growth inhibition was higher for MCF-7 cells than for MCF-10A cells. At 1 µM BisBAL NP, MCF-7 growth inhibition was 51%, while it was 11% for MCF-10A; at 25 µM BisBAL NP, the growth inhibition was 81% for MCF-7 and 24% for MCF-10A. With respect to mechanisms of action, a 24-hour exposure of 10 and 100 µM BisBAL NP caused loss of cell membrane integrity and fragmentation of tumor cell DNA. BisBAL NPs at 10 µM were genotoxic to and caused apoptosis of breast cancer cells. CONCLUSION: BisBAL NP-induced growth inhibition is dose dependent, and breast cancer cells are more vulnerable than noncancer breast cells. The mechanism of action of BisBAL NPs may include loss of plasma membrane integrity and a genotoxic effect on the genomic DNA of breast cancer cells.

Removal of internally deposited gold by 2,3-dimercaptopropane sodium sulphonate (Dimaval).

1 Orally administered 2,3-dimercaptopropane sodium sulphonate (DMPS, Dimaval) reduced the concentration of gold in rats treated with Auro-Detoxin and increased the urinary excretion of the metal. 2 In a long-term experiment, DMPS decreased significantly the concentration of gold in the kidneys and in the skin and increased it in plasma. 3 DMPS appears to be of interest as a possible antidote to gold, which could replace the more toxic 2,3-dimercaptopropanol (BAL).

FDA-preapproved drugs targeted to the translational regulation and processing of the amyloid precursor protein.

The 5' untranslated region (5'UTR) of the transcript encoding the Alzheimer's amyloid precursor protein (APP) is a key regulatory sequence that determines the amount of intracellular APP holoprotein present in brain derived cells. Using neuroblastoma cells (SY5Y) we developed a transfection based screen of a library of FDA drugs to identify compounds that limited APP luciferase reporter expression translated from the APP 5'UTR. Paroxetine (Paxil trade mark ), dimercaptopropanol, phenserine, desferrioxamine, tetrathiolmobdylate, and azithromycin were six leads that were subsequently found to also suppress APP holoprotein levels or to alter APP cleavage (azithromycin). Since APP holoprotein levels are proportionate to Abeta peptide output in many systems we tested the efficacy of paroxetine and dimercaptopropanol to limit Abeta secretion as measured by ELISA assays. Paroxetine and dimercaptopropanol limited Abeta peptide secretion from lens epithelial cells (B3 cells). Interestingly, paroxetine changed the steady-state levels of transferrin receptor mRNAs. These data suggested that this serotonin reuptake inhibitor (SSRI) provided extra pharmacological action to chelate interacellular iron or change the intracellular iron distribution. An altered iron distribution would be predicted to indirectly limit APP holoprotein expression and Abeta peptide secretion.

Properties of serum lipase in patients with various pancreatic diseases. Analysis by a new serum lipase assay method (the BALB-DTNB method) in combination with gel-filtration and iso-electrofocusing techniques.

Very low levels of lipase can easily be measured by a new serum lipase assay method (the BALB-DTNB method), using BAL-tributyrate (BALB) as a substrate, 5,5'-dithiobis(2-nitrobenzoic acid) as a chromogenic SH reagent, phenylmethylsulfonylfluoride as an inhibitor of esterases and sodium dodecyl sulfate as a surfactant. The BALB-DTNB method has a higher sensitivity than the conventional serum lipase assay methods, and proved useful for analyzing the properties of serum lipases in combination with gel-filtration on a Sephacryl S 200 column and isoelectrofocusing in an Ampholine column. Serum samples containing high levels of lipases from patients with pancreatic diseases or patients in whom the pancreatic exocrine gland had been stimulated by injecting caerulein and secretin were analyzed by these methods. The lipolytic profiles obtained indicated the presence of a lipase with an estimated molecular weight of 46,000 and isoelectric points of 7.4, 6.8, or/and 6.4. A lipase with properties similar to those of the serum lipase was found to be present in human pancreatic juice.

A novel and simple colorimetric assay for human serum lipase.

A new and simple colorimetric method for human serum lipase [EC 3.1.1.3] assay has been developed, using 2,3-dimercaptopropan-1-ol tributyroate as a substrate, 5,5'-dithiobis(2-nitro-benzoic acid) as a chromogenic reagent, phenylmethylsulfonyl fluoride as an inhibitor of serum esterases, and sodium dodecylsulfate as a lipase activator. The method requires only 50 micron1X2 of serum sample and a reaction time of less than 30 min. The method is reproducible and sensitive enough to measure low levels of lipase activity in normal and abnormal sera. The gel filtration of serum samples on a Sephadex G-200 column gave one peak of lipase activity, when measured by the present method, and the molecular weight of the enzyme was identical with that of lipase of human pancreatic origin, confirming the specificity of this new method for the serum lipase.

Biological chelation: 2,3-dimercapto-propanesulfonic acid and meso-dimercaptosuccinic acid.

Water soluble analogs of British Anti-Lewisite that are active orally and less toxic than BAL are now available. These agents are 2,3-dimercapto-1-propanesulfonic acid and meso-dimercaptosuccinic acid. Evidence for their effectiveness in preventing the lethal effects of sodium arsenite in mice and lewisite in rabbits is presented. These analogs can be expected to replace BAL in the treatment of heavy metal poisoning.

Hypochlorous acid-induced zinc release from thiolate bonds: a potential protective mechanism towards biomolecules oxidant damage during inflammation.

It has been proposed that metalloprotein zinc mobilization mediated by hypochlorous acid (HOCl) may induce cell injury (see H. Fliss and M. Ménard (1991), Archives of Biochemistry and Biophysics, 287, 175-179). In the present paper, we have demonstrated using a dimercaptopropanol-zinc complex that, once released from thiolate bonds by HOCl, zinc can exert a significant antioxidant effect on both linolenic acid and deoxyribose oxidation induced by iron. In these experimental conditions, however, the antagonism towards deoxyribose oxidation is notably less than that towards linolenic acid peroxidation, thus suggesting a more specific inhibitory effect of zinc on iron-mediated oxidant damage when polyunsaturated fatty acids represent the oxidizable substrate. The antioxidant effects of zinc are strictly related to the "free" form; indeed, the dimercaptopropanol-zinc complex per se is stimulatory even on biomolecules oxidant damage, apparently as a result of the prooxidant prooxidant interaction of the thiol compound with iron. In light of these results, it may be proposed that the zinc released from thiolate bonds by HOCl could specifically limit tissue oxidative burden in pathological conditions involving neutrophil accumulation and activation, such as inflammation and ischemia-reperfusion.

The excretion of trace elements in rat urine after treatment with 2,3-dimercaptopropane sodium sulfonate.

Administration of various doses of the chelating agent 2,3-dimercaptopropane sodium sulfonate (Dimaval, DMPS) leads to a greatly enhanced excretion of Zn and Cu. The excretion of Co, Mn, Ni and Fe remains unchanged. The relevance of these findings to the toxicity of DMPS is discussed.

Influence of 2,3-dimercaptopropane-1-sulfonate and dimercaptosuccinic acid on the mobilization of mercury from tissues of rats pretreated with mercuric chloride, phenylmercury acetate or mercury vapors.

The efficiency of the sodium salt of 2,3-dimercaptopropanesulfonic acid (DMPS) and meso-dimercaptosuccinic acid (DMSA) to mobilize mercury from tissues has been assessed in rats pretreated with different doses of HgCl2, phenylmercury acetate or exposed to different concentrations of mercury vapors. These pretreatments increase the mercury concentration in the kidney and to a lower extent in the liver. Only exposure to metallic mercury vapor leads to mercury accumulation in the brain. Both chelators mobilize mercury stored in the kidney and the amount of metal excreted in urine following a single administration of DMSA is a good indicator of the renal burden of mercury. The rate of removal is greater after DMPS administration than after DMSA but repeated administration of either agents eventually leads to the same total amount of mercury mobilized from the kidney. The loss of mercury from the liver can be slightly accelerated by repeated administration of the chelators. However, the chelators are inefficient in removing mercury from the brain.

Anti-lewisite activity and stability of meso-dimercaptosuccinic acid and 2,3-dimercapto-1-propanesulfonic acid.

Meso-dimercaptosuccinic acid (DMSA) and the sodium salt of 2,3-dimercapto-1-propanesulfonic acid (DMPS) are analogous in chemical structure to dimercaprol (BAL, British Anti-Lewisite). Dimercaprol was among the first therapeutically useful metal chelating agents and was developed originally as an anti-lewisite agent. Either DMSA or DMPS protects rabbits from the lethal systemic action of dichloro(2-chlorovinyl)arsine (29.7 mumols/kg, also known as lewisite. The analogs are active in this respect when given either sc or po. The stability of each of the three dimercapto compounds in distilled H2O, pH 7.0 at 24 degrees, has been examined for seven days. DMSA retained 82% of its mercapto groups, but no titratable mercapto groups remained in the DMPS or BAL solutions. At pH 5.0, however, there was no striking difference in the stability of the three dimercapto compounds (78-87%) over a seven day period. DMSA and DMPS warrant further investigation as water soluble metal binding agents in both in vivo and in vitro experiments.

Goldsalts, levamisole and D-penicillamine as first choice slow-acting antirheumatic drugs in rheumatoid arthritis--a long-term follow-up study.

In the present study, 345 rheumatoid arthritis patients were treated using goldsalts, D-Penicillamine or levamisole as the slow-acting antirheumatic drug of first choice. Goldsalts were given to 182 patients, levamisole to 139 and D-Penicillamine to 24. At the time of the present evaluation, 83 patients were still on goldsalts (44.6%), 63 on levamisole (45.2%) and 11 on D-Penicillamine (45.9%). Adverse reactions required interruption of treatment in 64 patients on goldsalts (35.2%), in 44 on levamisole (31.7%) and in 5 on D-Penicillamine (20.8%). Inefficacy was responsible for withdrawal of 33 patients receiving goldsalts (18.1%), 30 receiving levamisole (21.6%) and 8 receiving D-Penicillamine (33.3%). The duration of treatment was 4.6 years for goldsalts, 3.6 years for levamisole and 3.6 years for D-Penicillamine. In the present analysis none of the compounds was found to have a definite advantage over the others. The rather favourable treatment continuation rates in this study can be attributed to the fact that the slow-acting antirheumatic drugs were given at an early stage of the disease, preferably before the occurrence of radiological lesions.