Uptake, toxicity and radiation effects of the boron compounds DAAC-1 and DAC-1 in cultured human glioma cells.

PURPOSE: To study the uptake, toxicity and radiation effects in vitro of a diol-amino acid-carborane (DAAC-1) and make comparisons with the previously studied diol-amine-carborane (DAC-1). MATERIALS AND METHODS: Toxicity and radiation effects were studied with clonogenic survival, uptake by measuring the cellular boron content and the subcellular distribution was investigated after organelle separation with centrifugation. The studied cell line was human glioma U343. RESULTS: DAAC-1 showed an accumulation of 1-1.5 times, compared with the culture medium, and was non-toxic up to 47 microg boron/ml. The accumulation of DAC-1 was about 90 times, but toxic effects were detectable already at the concentration 5 microg boron/ml. None of the compounds was localized in the cell nucleus. Following irradiation with thermal neutrons, DAC-1 was about 2.5 times more effective than DAAC-1 and about 4.9 times more effective than neutrons alone, at the survival level 0.2. The dose modifying factors, when compared with the neutron beam alone, were for both DAAC-1 and DAC-1 about 1.5 and about 5 when compared with 60Co-gamma-radiation. CONCLUSIONS: DAAC-1 was less toxic than DAC-1 but gave less accumulation of boron. Both substances gave significant boron-dependent cell inactivation when the test cells were exposed to thermal neutrons.

Chemistry and biology of some low molecular weight boron compounds for boron neutron capture therapy.

Boronated DNA targeting agents are especially attractive candidates for BNCT because they may deliver boron-10 to the nuclei of tumor cells. Numerous boron-containing analogs have been synthesized and some have shown promising results in initial biological tests. One of the most challenging tasks in this special field of research remains the finding of suitable targeting strategies for the selective delivery of boron rich DNA-intercalator/alkylator to tumor cells. Synthetic and biological studies of boron compounds suitable for DNA-binding are reviewed. The amino acid p-boronophenylalanine (BPA) is presently of considerable clinical interest. Other boronated amino acids might also be candidates for BNCT either per se, as part of part of tumor-seeking peptides or conjugated to targeting macromolecules. A large number of boronated L- and D-amino acids with varying liphophicility and sterical requirements are now available for evaluation. Recent synthetic and biological studies of aromatic boronoamino acids, carboranylamino acids and carboranyl amines are also reviewed.

In vitro determination of toxicity, binding, retention, subcellular distribution and biological efficacy of the boron neutron capture agent DAC-1.

In boron neutron capture therapy (BNCT), 10B is delivered selectively to the tumour cells and the nuclide then forms high-LET radiation (4He2+ and 7Li3+) upon neutron capture. Today much research is focused on development of a variety of boron compounds aimed for BNCT. The compounds must be thoroughly analysed in preclinical tests regarding basic characteristics such as binding and subcellular distribution to enable accurate estimations of dose-modifying factors. DAC-1,2-[2-(3-amino-propyl)-1,2-dicarba-closo-dodecaboran (12)-1-yl-methoxy]- 1,3-propanediol was synthesized at our laboratories and the human colon carcinoma cells LS-174T were used as an in vitro model. The boron compound showed a remarkable intracellular accumulation, 20-100 times higher than the boron content in the culture medium, in cultured cells and was not removed by extensive washes. Approximately half of the boron taken up also remained within the cells for at least 4 days. The DAC-1 compound alone was not toxic at boron concentrations below 2.5 micrograms B/g. The intracellular distribution of the boron compound was investigated by subcellular fractionation experiments and low pH treatments. It is possible that DAC-1 binds to some intracellular molecules or to membranes connected with organelles in the cytoplasm or even to the inside of the outer cell membrane. Another possibility is that the compound, due to the somewhat lipophilic properties, is embedded in the membranes. Thermal neutron irradiations were carried out at the Brookhaven Medical Research Reactor (BMRR). At a survival level of 0.1, DAC-1 + thermal neutrons were about 10.5 times more effective in cell inactivation than the thermal neutrons alone. Monte Carlo calculations gave a mean value of the 10B-dependent specific energy, the dose, of 0.22 Gy. The total physical dose during irradiation of DAC-1-containing cells with a neutron fluence of 0.18 x 10(12) n/cm2 was 0.39 Gy. The dose-modifying factor, at survival level 0.1, when comparing irradiation with thermal neutrons with and without DAC-1 was 3.4, while the dose-modifying factor when comparing neutron irradiations of cells with DAC-1 and irradiation of the cells with 60Co-gamma was 7.3. The results are encouraging and in vivo tests of tissue distributions and tumour uptake should now be carried out.

Pharyngo-oesophageal webs in dysphageal patients. A radiologic and clinical investigation in 1134 patients.

Among 1134 patients, cineradiologically examined because of dysphagia, 85 (7.5%) had webs in the pharyngo-oesophageal segment. Webs were more common in women (10%) compared to men (5%). Radiologic characteristics of the webs such as precise location, multiplicity, circumferential extension, thickness, accompanying streamline phenomenon and encroachment on the lumen, were compared to the presence of concomitant anaemia, thyroid disease, neoplasm, as well as the age and sex of the patients. Webs were regularly deeper in women compared to men. Patients with iron deficiency anaemia had thicker webs compared to patients without such anaemia. No other radiologic characteristics were found that could be used for distinguishing these potentially more significant webs from those in patients without such concomitant diseases.

Cyclic AMP accumulation in human fibroblast cultures: diabetics compared with normals.

Isoproterenol-stimulated accumulation of adenosine 3',5'-monophosphate (cyclic AMP) in skin fibroblast cultures from type 2 diabetics was twice as high as in fibroblasts from control subjects.

Effect of LDL from smokers and non-smokers on cell mass, proliferation, and collagen secretion by rabbit smooth muscle cells and human skin fibroblasts in culture.

The effects of low density lipoproteins (LDL) from non-smokers and heavy smokers on rabbit aortic smooth muscle cells and human skin fibroblasts in culture were studied. The addition of LDL in increasing concentrations resulted in a decrease in cell mass. When data correction for cell loss was made, collagen secretion and cell proliferation were found to have been increased by LDL. The two different cell types used did not differ in their response to LDL. The effect of LDL from smokers and non-smokers was the same. Thus there was no indication of growth stimulation by factors carried by LDL from smokers, i.e. lipid-soluble hydro-carbons--mutagens.

Plasma insulin and C-peptide in relation to glucose intolerance in middle-aged men.

The relation between glucose homeostasis and insulin secretion (immunoreactive insulin and C-peptide) was studied in middle-aged males matched for age and body weight. Subjects with mild type II diabetes mellitus were compared to normals and to individuals with impaired glucose tolerance (IGT). In addition, the diabetics were subdivided according to duration, some of the subjects having recently deteriorated from IGT status. In the IGT individuals, there were no indications of a reduction in basal or glucose-induced insulin output. On the contrary, data indicate somewhat higher than normal secretion. Within the type II diabetics, those of short duration were largely similar to normals, whereas diabetes of longer duration was associated with some diminution in indices of B cell secretion. The data support the notion that a deficient insulin output is not a primary pathophysiological event in the development of type II diabetes.

Plasma cyclic nucleotides and plasma catecholamines before and after prolonged treatment with clonidine in hypertensive patients.

The effect of standing and physical exercise and catecholamines and cyclic nucleotides in plasma was measured in 8 patients with essential hypertension under standardized conditions before and after prolonged treatment with clonidine. Before clonidine medication noradrenaline, adrenaline and cyclic AMP (cAMP) increased in response to standing and bicycling for 20 min. No significant correlation was found between their absolute levels nor was the increase in cAMP following exercise correlated to the increase in noradrenaline. Standing and physical exercise were without effect on cyclic GMP (cGMP). Clonidine reduced the plasma noradrenaline concentration in supine position and the noradrenaline and the adrenaline response to standing and exercise. Plasma cAMP was uneffected by clonidine under basal conditions but the response to exercise was slightly reduced initially. During clonidine there was a positive correlation between the plasma levels of cAMP and noradrenaline following work. Clonidine produced an increase in plasma cGMP in supine position, immediately prior to bicycling and after 5 min of exercise.

Plasma insulin and C-peptide in normal and glucose intolerant males: the role of hepatic insulin uptake.

Plasma immunoreactive insulin and peptide were measured in the basal and glucose-stimulated state in middle-age males. Normals were compared with individuals with mild to moderate glucose intolerance. Ths results demonstrate the value of C-peptide measurements in the assessment of "true" insulin secretion and suggest that a reduced hepatic insulin extraction is a feature of the glucose intolerant state.

Finger clubbing and aspartylglucosamine excretion in a laxative-abusing patient.

A young woman with a previous history of anorexia nervosa presented with severe finger clubbing. Urine samples intermittently contained significant amounts of aspartylglucosamine. Liver biopsy showed abnormal cytoplasmic inclusions in phagocytic cells. The patient was found to be abusing senna laxative.

Adenylate cyclase activity in human liver membranes and its inhibition by adenosine and adenine nucleotides.

The production of adenosine 3',5'-monophosphate (cyclic AMP) in a membrane preparation from human liver homogenate has been studied. Cyclic AMP production was enhanced by glucagon, guanylyl 5'-imidodiphosphate (GMP-PNP), or fluoride, or combinations of these. Adenosine, adenosine monophosphate (AMP) and adenosine diphosphate (ADP) at a concentration of 10(-3) mol/l antagonized the effects of all stimulants. These data suggest that inhibitory effects are exercised at the catalytic moiety of the adenylate cyclase system, or at the transducer function between hormone receptor and catalytic unit. In contrast, adenosine at a concentration of 10(-5) mol/l antagonized glucagon- but not fluoride-stimulated adenylate cyclase activity.

Glucagon effects on plasma cyclic AMP and other reactants in normals and low insulin responders.

The metabolic response to i.v. glucagon was evaluated in 11 normal individuals and 8 healthy low insulin responders. Elevations of plasma cyclic AMP and blood glucose were similar in both groups. Accordingly, no indications were seen of differing hepatic responsiveness to glucagon. In contrast, the groups differed in the course of plasma glycerol during the test.

Urinary cyclic AMP: relation to calcium balance and comparison of assay methods.

Urinary adenosine 3',5'-monophosphate (cyclic AMP, cAMP) has been determined in 84 males. Two protein-binding assays were used: the method of Gilman and the Amersham assay kit. The results were in close agreement. The excretion of cAMP was nor correlated to urinary calcium or to estimated calcium intake.

Plasma myeloperoxidase and lactoferrin measured by radioimmunoassay: relations to neutrophil kinetics.

In 31 patients, covering a wide range of blood neutrophil counts and turnover rates, the plasma concentrations of myeloperoxidase and lactoferrin have been measured with radioimmunoassays and compared to neutrophil kinetic parameters, measured with DF32P-labeled neutrophils. It was found that the plasma concentrations of both proteins correlated significantly with the total number of neutrophils in the blood (TBGP=total blood granulocyte pool) as well as with the neutrophil turnover rate (GTR=granulocyte turnover rate), which is evidence that neutrophilic granulocytes are the main suppliers of myeloperoxidase and lactoferrin to the plasma. In contrast to the previously demonstrated better relationship between the GTR and plasma lysozyme, a protein also originating in neutrophil granules, both myeloperoxidase and lactoferrin correlated better with the TBGP. These differences may reflect differences in the mode of release of intragranular proteins from neutrophils to the plasma. The correlation of the plasma lactoferrin concentration with the TBGP was so good as to suggest its use in the clinical assessment of the TBGP.