Boron concentration measurement in biological tissues by charged particle spectrometry

Measurement of boron concentration in biological tissues is a fundamental aspect of boron neutron capture therapy, because the outcome of the therapy depends on the distribution of boron at a cellular level, besides on its overall concentration. This work describes a measurement technique based on the spectroscopy of the charged particles emitted in the reaction ¹⁰B(n,α)⁷Li induced by thermal neutrons, allowing for a quantitative determination of the boron concentration in the different components that may be simultaneously present in a tissue sample, such as healthy cells, tumor cells and necrotic cells. Thin sections of tissue containing ¹⁰B are cut at low temperatures and irradiated under vacuum in a thermal neutron field. The charged particles arising from the sample during the irradiation are collected by a thin silicon detector, and their spectrum is used to determine boron concentration through relatively easy calculations. The advantages and disadvantages of this technique are here described, and validation of the method using tissue standards with known boron concentrations is presented.     

The interaction between salinity and boron toxicity affects the subcellular distribution of ions and proteins in wheat leaves

Salinity aggravates B toxicity symptoms in several plant species. In the present study the interactive effects of B toxicity and salinity stresses on the subcellular distribution of boron, cations and proteins in basal and apical leaf sections of wheat were investigated. High B supply increased total B concentrations in all leaf parts, but values remained below 25 mg B kg^?1 dry weight (DW) in basal sections, whereas they reached more than 600 mg B kg^?1 DW in leaf tips. In basal leaf sections intercellular soluble B concentrations closely reflected the external supply, whereas intracellular soluble B concentrations remained lower by a factor of two, indicating some retention of excess B in the apoplast. Combined salinity and B toxicity stresses significantly increased soluble B concentrations in inter‐ and intracellular compartments of basal leaf sections in comparison with either stress alone, probably related to salinity‐induced changes in water status. The combined stresses also induced quantitative and qualitative changes in inter‐, but not intracellular protein composition. Most obvious was the induction of a 25 kDa protein and an increase in amount of a 33 kDa protein. It is suggested that these changes might be due to structural modifications of the cell wall. The concentration of soluble boron in cells is proposed to be an indicator of boron toxicity.     

Quantitative subcellular secondary ion mass spectrometry (SIMS) imaging of boron-10 and boron-11 isotopes in the same cell delivered by two combined BNCT drugs: in vitro studies on human glioblastoma T98G cells

Ion microscopy was used for subcellular quantitative imaging of the isotopes 10B and 11B in the same cell to evaluate boron delivery using a mixture of two neutron capture therapy drugs, p-boronophenylalanine-fructose (BPA-F) and sodium borocaptate (BSH). The application of 10B-labeled BPA-F and 11B-labeled BSH allowed independent imaging of both 10B and 11B in the same cell using a CAMECA IMS-3f ion microscope. Mixed-drug treatments were compared to single-drug exposures given under identical conditions. 10BPA-F delivered 10B heterogeneously to T98G human glioblastoma cells, with a significantly reduced concentration in an organelle-rich perinuclear region. The intracellular distribution of 11B from 11BSH contrasted with that of the 10B from 10BPA-F, with 11B distributed nearly homogeneously throughout cells. The subcellular distributions of 10B and 11B were sustained in mixed-drug treatments and resembled their localizations after the single-drug treatments. In both single- and mixed-drug treatments, cellular levels of 10B from 10BPA-F nearly doubled between 1 h and 6 h, with a 3:1 intracellular to nutrient medium partitioning, while cellular levels of 11BSH remained essentially unchanged. The net effect of the combined treatment with 10BPA-F and 11BSH was an additive delivery of boron to cells. This study introduces a novel approach for checking potential synergistic, antagonistic or simple additive delivery of two mixed boronated compounds in cellular/subcellular compartments.     

Use of ex vivo binding to measure the brain concentrations of putative radioligands

The development of radioligands capable of imaging brain receptors depends on, amongst other factors, the ability of such compounds to penetrate the blood-brain barrier. We describe an ex vivo binding technique for measuring the brain concentration of peripherally administered unlabeled compounds. This technique can be used early in the development of putative radioligands. The pharmacokinetics of brain penetration of three muscarinic antagonists are described: QNB, BrQNB and the 2-thienyl derivative of BrQNB and were found to compare favorably to previous studies using [³H]QNB. These studies demonstrate the effectiveness of ex vivo binding in assessing the brain concentration of peripherally administered unlabeled compounds.     

Effects of boron derivatives on extracellular matrix formation

Boric acid solution (3%) dramatically improves wound healing through action on the extracellular matrix, a finding that has been obtained in vitro. Consequently, investigations are presently underway to produce boronated compounds having a therapeutical effectiveness similar to that of boric acid. On the basis of experimental results obtained with boric acid, we examined the effects of boron derivatives on extracellular matrix formation and degradation and analyzed their potential toxicity by using two biological models (chick embryo cartilage and human fibroblasts). The four boron derivatives tested in this study (triethanolamine borate; N-diethyl-phosphoramidate-propylboronique acid; 2,2 dimethylhexyl-1,3-propanediol-aminopropylboronate and 1,2 propanediol-aminopropylboronate) mimicked the effects of boric acid. They induced a decrease of intracellular concentrations in extracellular matrix macromolecules (proteoglycans, proteins)-associated with an increase of their release in culture medium and stimulated the activity of intra- and extracellular proteases. Similarly to boric acid, these actions occurred after exposure of the cells to concentrations of all boron derivatives without apparent toxic effects. The compounds were found to be more toxic than boric acid itself when concentrations were calculated according to their molecular weight. Nevertheless, these in vitro preliminary results demonstrate effects of boron derivatives that may be of therapeutic benefit in wound repair.     

Boron uptake in normal melanocytes and melanoma cells and boron biodistribution study in mice bearing B16F10 melanoma for boron neutron capture therapy

Information on (10)B distribution in normal tissues is crucial to any further development of boron neutron capture therapy (BNCT). The goal of this study was to investigate the in vitro and in vivo boron biodistribution in B16F10 murine melanoma and normal tissues as a model for human melanoma treatment by a simple and rapid colorimetric method, which was validated by HR-ICP-MS. The B16F10 melanoma cell line showed higher melanin content than human melanocytes, demonstrating a greater potential for boronophenylalanine uptake. The melanocytes showed a moderate viability decrease in the first few minutes after BNCT application, stabilizing after 75 min, whereas the B16F10 melanoma showed the greatest intracellular boron concentration at 150 min after application, indicating a different boron uptake of melanoma cells compared to normal melanocytes. Moreover, at this time, the increase in boron uptake in melanoma cells was approximately 1.6 times higher than that in normal melanocytes. The (10)B concentration in the blood of mice bearing B16F10 melanoma increased until 90 min after BNCT application and then decreased after 120 min, and remained low until the 240th minute. On the other hand, the (10)B concentration in tumors was increased from 90 min and maximal at 150 min after application, thus confirming the in vitro results. Therefore, the present in vitro and in vivo study of (10)B uptake in normal and tumor cells revealed important data that could enable BNCT to be possibly used as a treatment for melanoma, a chemoresistant cancer associated with high mortality.     

Saccharomyces cerevisiae Bor1p is a boron exporter and a key determinant of boron tolerance

Boron is toxic to living organisms when present in excess. Saccharomyces cerevisiae Bor1p is a plasma membrane protein that decreases the intracellular concentration of boron and confers boron tolerance in yeasts. We investigated the detailed characteristics of boron transport by Bor1p and its roles in boron tolerance. Boron transport assays showed that the bor1 deletion mutant (bor1Delta) accumulates higher intracellular concentrations of boron and has a lower rate of boron export. The bor1Delta showed greater susceptibility to high concentrations of boron than the wild-type strain, and the growth rates of both strains were negatively correlated with the intracellular concentrations of boron. With normal to toxic levels of external boron, green fluorescent protein (GFP)-tagged Bor1p localized to the plasma membrane irrespective of the concentration of boron in the medium. Taken together, these results establish Bor1p as a plasma membrane boron exporter and a key determinant of boron tolerance.     

Analysis of boron distribution in vivo for boron neutron capture therapy using two different boron compounds by secondary ion mass spectrometry

The efficiency of boron neutron capture therapy (BNCT) for malignant gliomas depends on the selective and absolute accumulation of (10)B atoms in tumor tissues. Only two boron compounds, BPA and BSH, currently can be used clinically. However, the detailed distributions of these compounds have not been determined. Here we used secondary ion mass spectrometry (SIMS) to determine the histological distribution of (10)B atoms derived from the boron compounds BSH and BPA. C6 tumor-bearing rats were given 500 mg/kg of BPA or 100 mg/kg of BSH intraperitoneally; 2.5 h later, their brains were sectioned and subjected to SIMS. In the main tumor mass, BPA accumulated heterogeneously, while BSH accumulated homogeneously. In the peritumoral area, both BPA and BSH accumulated measurably. Interestingly, in this area, BSH accumulated distinctively in a diffuse manner even 800 microm distant from the interface between the main tumor and normal brain. In the contralateral brain, BPA accumulated measurably, while BSH did not. In conclusion, both BPA and BSH each have advantages and disadvantages. These compounds are considered to be essential as boron delivery agents independently for clinical BNCT. There is some rationale for the simultaneous use of both compounds in clinical BNCT for malignant gliomas.     

Manipulation of in Vivo Sorbitol Production Alters Boron Uptake and Transport in Tobacco

Recent evidence that some species can retranslocate boron as complexes with sugar alcohols in the phloem suggests a possible mechanism for enhancing boron efficiency. We investigated the relationship between sugar alcohol (sorbitol) content, boron uptake and distribution, and translocation of foliar-applied, isotopically enriched ¹⁰B in three lines of tobacco (Nicotiana tabacum) plants differing in sorbitol production. In tobacco line S11, transformed with sorbitol-6-phosphate dehydrogenase, the production of sorbitol was accompanied by an increase in the concentration of boron in plant tissues and an increased uptake of boron compared with either tobacco line A4, transformed with antisense orientation of sorbitol-6-phosphate dehydrogenase, or wild-type tobacco (line SR1, zero-sorbitol producer). Foliar application of ¹⁰B to mature leaves was translocated to the meristematic tissues only in line S11. These results demonstrate that the concentration of the boron-complexing sugar alcohol in the plant tissue has a significant effect on boron uptake and distribution in plants, whereas the translocation of the foliar-applied ¹⁰B from the mature leaves to the meristematic tissues verifies that boron is mobile in sorbitol-producing plants (S11) as we reported previously. This suggests that selection or transgenic generation of cultivars with an increased sugar alcohol content can result in increased boron uptake, with no apparent negative effects on short-term growth.     

Factors controlling equilibrium boron (B) concentration in nutrient solution buffered with B-specific resin (Amberlite IRA-743)

In conventional solution culture where boron (B) is added as boric acid, fluctuating external B supply often produces confounding and ill-defined physiological and biochemical responses in plants, especially when grown at deficient and marginal B supply. Our previous studies proposed the use of the B-specific resin – Amberlite IRA-743 to develop a B-buffered solution culture. The present study aims to evaluate crucial factors determining equilibrium B concentrations in nutrient solution buffered with the B-loaded resin, including the B loading of the resin, pH in the nutrient solution and B removal from the solution. The equilibrium B concentrations in nutrient solution were determined by both the amount of B sorbed by the resin and the solution pH. At pH 6.05±0.05, the relationship between the resin B content and equilibrium B concentration in the nutrient solution is closely described by the equation: Y = 18.8 X^1.457 [ where, Y = equilibrium B concentration (μM) in nutrient solution and X = B content of the resin (mg B g⁻¹ moist resin)]. However, at a given resin B content, lowering solution pH from 7 to 4 significantly increased B concentrations in solution through the release of B from the solid phase of the resin beads. The B-loaded resin was capable of maintaining stable B concentrations in the nutrient solutions, ranging from deficient to marginally adequate B concentrations for dicot species. In conclusion, B concentrations ranging from 0.05 to 11 μM, were buffered for 5 days with the resin loaded with 0.004 – 0.691 mg B g⁻¹ moist resin in the nutrient solution. Precise pH control in the nutrient solution is critical for the success of a B-buffered solution culture study. This revised version was published online in June 2006 with corrections to the Cover Date.     

Roles of BOR1, DUR3, and FPS1 in boron transport and tolerance in Saccharomyces cerevisiae

The roles of three membrane proteins, BOR1, DUR3, and FPS1, in boron (B) transport in yeast were examined. The boron concentration in yeast cells lacking BOR1 was elevated upon exposure to 90 mM boric acid, whereas cells lacking DUR3 or FPS1 showed lower boron concentrations. Compared with control cells, cells overexpressing BOR1 or FPS1 had a lower boron concentration, and cells overexpressing DUR3 had a higher boron concentration. These results suggest that, in addition to the efflux boron transporter BOR1, DUR3 and FPS1 play important roles in regulating the cellular boron concentration. Analysis of the yeast transformants for tolerance to a high boric acid concentration revealed an apparent negative correlation between the protoplasmic boron concentration and the degree of tolerance to a high external boron concentration. Thus, BOR1, DUR3, and FPS1 appear to be involved in tolerance to boric acid and the maintenance of the protoplasmic boron concentration.     

Biodistribution of sodium borocaptate (BSH) for boron neutron capture therapy (BNCT) in an oral cancer model

Boron neutron capture therapy (BNCT) is based on selective accumulation of ¹⁰B carriers in tumor followed by neutron irradiation. We previously proved the therapeutic success of BNCT mediated by the boron compounds boronophenylalanine and sodium decahydrodecaborate (GB-10) in the hamster cheek pouch oral cancer model. Based on the clinical relevance of the boron carrier sodium borocaptate (BSH) and the knowledge that the most effective way to optimize BNCT is to improve tumor boron targeting, the specific aim of this study was to perform biodistribution studies of BSH in the hamster cheek pouch oral cancer model and evaluate the feasibility of BNCT mediated by BSH at nuclear reactor RA-3. The general aim of these studies is to contribute to the knowledge of BNCT radiobiology and optimize BNCT for head and neck cancer. Sodium borocaptate (50 mg ¹⁰B/kg) was administered to tumor-bearing hamsters. Groups of 3–5 animals were killed humanely at nine time-points, 3–12 h post-administration. Samples of blood, tumor, precancerous pouch tissue, normal pouch tissue and other clinically relevant normal tissues were processed for boron measurement by optic emission spectroscopy. Tumor boron concentration peaked to therapeutically useful boron concentration values of 24–35 ppm. The boron concentration ratio tumor/normal pouch tissue ranged from 1.1 to 1.8. Pharmacokinetic curves showed that the optimum interval between BSH administration and neutron irradiation was 7–11 h. It is concluded that BNCT mediated by BSH at nuclear reactor RA-3 would be feasible.     

Distribution and Partitioning of Newly Taken-up Boron in Sunflower

Distribution and partitioning of newly acquired boron (B) in a mature sunflower (Helianthus annuus L., cv. 3101) plant was investigated. In leaf blades of sunflower plants grown under 0.93, 2.8, and 9.3 mmol B m⁻³, the level of cell-wall-bound B was rather uniform, irrespective of leaf position and B concentration. Boron concentration gradients among leaf positions were produced mainly by different levels of water-soluble B. To determine the distribution of newly taken-up B in plant parts, ¹⁰B-labeled boric acid at a concentration of 2.8 mmol B m⁻³ was applied. The majority of newly acquired B was delivered to the younger leaves, however, approximately one-fourth of the B in the top and second leaves was the older B which was taken up before the 6 d treatment period. In the root tissues, two-fifth of the water-soluble B was new B taken up in the last 6 d, however, within 6 h of the application new B contributed to approximately 80% of the xylem sap B, suggesting that newly taken-up B is preferentially transported to the shoots. When B was withdrawn from the culture solution, the B concentration per leaf area of the lower leaves decreased slightly over 9 d. However, there was an abrupt decrease in the younger leaves, even when taking into account the rapid expansion of the leaf blade, suggesting that B moves more rapidly from the younger leaves than from the older leaves     

Cat Heart Muscle in Vitro : III. The extracellular space

The "osmotic gradient" method, an intracellular microelectrode technique for determining whether an uncharged, water-soluble molecule enters cells or remains extracellular, is described. Using this method, a series of carbohydrates of graded molecular size were examined. In cat papillary muscles mannitol, molecular radius 4.0 Å, remained extracellular while arabinose, molecular radius 3.5 Å entered the cells. Measurement of the simultaneous uptake of H³-mannitol and C¹⁴-inulin showed that mannitol equilibrates with 40 per cent of total water in 1 hour, after which the mannitol space does not further increase. By contrast, inulin, molecular radius ~15 Å, equilibrates with 24 per cent of total water in 1 hour; thereafter the inulin space continues to increase very slowly. The intracellular K concentrations are significantly higher and the intracellular Na and Cl concentrations significantly lower when mannitol rather than inulin is used to measure the extracellular space. The intracellular Cl concentration determined with Cl³⁶ or Br⁸² is significantly higher than that calculated from the membrane potential assuming a passive Cl distribution. In addition, it is shown that choline enters and is probably metabolized by the cells of papillary muscle.     

Determination of the concentration of complex boronated compounds in biological tissues by inductively coupled plasma atomic emission spectrometry

The application of inductively coupled plasma atomic emission spectrometry (ICP-AES) to the determination of the concentration of complex boron-containing compounds in biological tissue samples is described. Tissue digestion is achieved with perchloric acid and hydrogen peroxide in 1 hr at 75 degrees C. The ICP-AES method gave a linear response for elemental boron concentration in the range 0.05 to 100 ppm and does not require the reduction of the boron to a simple species, such as boric acid. Complete recovery of boron in complex boron cluster compounds was obtained. The procedure has been applied to the determination of the boron content in compounds synthesised for neutron capture therapy and is suitable for use in biodistribution studies of such compounds.     

Cobalt bis(dicarbollide) versus closo-dodecaborate in boronated chlorin e(6) conjugates: implications for photodynamic and boron-neutron capture therapy

Conjugation of boron nanoparticles with porphyrins is an attractive way to create dual agents for anticancer boron neutron capture therapy (BNCT) and photodynamic therapy (PDT). Properties of chlorin e(6) conjugated with two cobalt bis(dicarbollide) nanoparticles (1) or with a closo-dodecaborate nanoparticle (2) are reported. Fluorescent dianionic conjugates 1 and 2 penetrate in A549 human lung adenocarcinoma cells, stain cytoplasm diffusely and accumulate highly in lysosomes but are not toxic themselves for cells. Average cytoplasmic concentration of boron atoms (B) achieves 270 μM (ca. 2 × 10(8) B/cell) and 27 μM (ca. 2 × 10(7) B/cell) at the 1.5 μM extracellular concentration of 1 and 2, respectively, that makes conjugate 1 especially suitable for BNCT. Conjugate 2 causes photoinduced cell death at micromolar concentrations and can be considered also as a photosensitizer for PDT. Conjugates 1 and 2 have high quantum yields of singlet oxygen generation (0.55 and 0.85 in solution, respectively), identical intracellular localization and similar lipid-like microenvironment but conjugate 1 possesses no photoinduced cytotoxicity. A presence of cobalt complexes in conjugate 1 is supposed to be a reason of the observed antioxidative effect in cellular environment, but an exact mechanism of this intriguing phenomenon is unclear. Due to increased intracellular accumulation and absence of photoinduced cytotoxicity conjugate 1 is promising for fluorescence diagnostics of cancer.     

The Mechanism of Boron Immobility in Plants

A mechanism is postulated to explain the immobility of boron in plants, i.e., the absence of any significant retranslocation of this element from one leaf to another. It is shown that boron readily enters the bark and is translocated within the bark. Since it has also been shown that boron remains water-soluble in plants, the immobility cannot be explained through a chemical fixation, lack of entry into phloem, or absence of phloem transport. Rather, boron enters the phloem in leaf margins where concentrations are high, is transported in these conduits, is lost therefrom where the xylem concentration is low, i.e. in basal areas of the leaf and in petiole, is transported back in the xylem and accumulates in terminal place of the transpiration stream. A high local mobility of boron, together with the essentially unidirectional flow of the transpiration stream, thus cause a cyclic movement of B and prevent the efflux of this nutrient; this explains the immobility over long distances.     

Ortho-carboranyl glycosides for the treatment of cancer by boron neutron capture therapy.

Distinct biological properties of the ortho-carboranyl (1,2-dicarba-closo-dodecaboranyl) glycosides 1, 2 and 3 were evaluated to estimate the suitability of these compounds for cancer treatment by boron neutron capture therapy. The boron uptake into B16-Melanoma cells was significantly higher by incubating the cells with aqueous solutions of carboranyl glucoside 1 (11.2 ppm after 3h), lactoside 2 (13.2 ppm after 12h) and maltoside 3 (20.0 ppm after 24h) compared with solutions of clinically used p-boronophenylalanine (BPA) 5 (3.1 ppm after 24h). Carboranyl maltoside 3 was more effective than boron-10 enriched 5 in killing C-6 rat glioma cells by incubating the cells with the compound and subsequent treatment with thermal neutrons. 3 was also administrated iv, in concentrations of 25 mg boron/kg body weight to rats bearing brain tumors. After a period of 4h after administration the concentration of boron in the tumor tissue was 3.0 ppm.     

Equinocandinas: aspectos aplicados de la farmacología

The echinocandins share pharmacodynamic properties, although there are some interesting differences in their pharmacokinetic behaviour in the clinical practice. They are not absorbed by the oral route. They have a somewhat special distribution in the organism, as some of them can reach high intracellular concentrations while, with some others, the concentration is reduced. They are highly bound to plasma proteins, thus it is recommended to administer a loading dose for anidulafungin and caspofungin, although this procedure is not yet clear with micafungin. Echinocandins are excreted via a non-microsomal metabolism, so the urinary concentration is very low. Some carrier proteins that take part in the biliary clearance process are probably involved in the interactions described with caspofungin and micafungin. These two drugs must be used with caution in patients with severely impaired hepatic function, while all of them can be used without special precautions when there is renal impairment or the patient requires renal replacement therapy.