Irregular variations in radiation sensitivity when the linear energy transfer is increased.

Seven cell lines were analyzed for clonogenic survival after irradiation with photons (60Co) or accelerated helium or nitrogen ions. The cell lines showed different sensitivity to photon radiation and most of the differences decreased after irradiation with helium ions with a linear energy transfer (LET) of about 40 keV/microns. However, all cell types had individual LET sensitization patterns and the mean relative biological effectiveness (RBE) at 10% survival ranged from 1.46 +/- 0.12 to 2.41 +/- 0.26 for the helium ions. This difference was significant and the differences increased further when higher survival levels were considered. There was only a weak tendency towards a relation between photon and helium ion sensitivity when the linear component of the survival curves, the alpha-values, were compared, and no relation at all for other parameters. It was not possible to predict the response to an increased LET from the photon responses obtained. Three of the cell lines were also irradiated with nitrogen ions with an LET of 125 keV/microns. These cells were, as expected, sensitized further and the average RBE at 10% survival was 3.67 +/- 0.67. However, one cell line was more resistant than the others in this case. Furthermore, the quadratic component of the survival curves, the beta values, were higher after irradiation with nitrogen than with helium ions. Thus, several irregular and unexpected results were seen when the LET was increased.

Cellular binding of carboranylalanine and some effects of boron neutron capture. Analysis of cultured melanoma B16 cells.

The boron containing substances L- and D-carboranylalanine might be of interest for boron neutron capture therapy, BNCT. Cultured mouse melanoma B16 cells were analyzed regarding binding of these substances and some introductory studies on effects of thermal neutron irradiation were also carried out. Comparisons were made with two boron containing compounds, p-boronophenylalanine (BPA) and boronated thiouracil (BTU-1), previously proposed for BNCT of melanomas. The results showed that both L- and D-carboranylalanine bound well in the B16 cells whereas BTU-1 gave no, and BPA only a low, binding. Thus, both forms of carboranes bound better than the two previously proposed substances. The carboranes also bound rather well in two tested human melanoma cell lines, IGR1 and RPMI-7951. Both L- and D-carboranylalanine showed a certain binding to isolated melanin but were not incorporated during melanin synthesis. Cultured glioma cells, used for comparison, bound BPA and to some extent the carboranes. This indicates that the substances are not melanoma specific. The carboranes caused some acute detachment of monolayer growing cells but were not strongly toxic since they did not reduce the growth rate. The cells treated with L-carboranylalanine or BPA showed, after neutron irradiation, a clear decrease in survival compared to the controls whereas no or only small effects were seen for cells treated with D-carboranylalanine or BTU-1. These results were conflicting since BPA gave therapeutical effects although only small amounts were bound while D-carboranylalanine gave no significant therapeutical effect in spite of better binding. One explanation might be different intracellular localizations. This has to be studied in more detail.

Penetration and binding of L- and D-carboranylalanine in human melanoma spheroids.

The principle of boron neutron capture therapy (BNCT) is that a cell-specific 10B-containing substance binds to tumour cells and irradiation with thermal neutrons is performed when the 10B concentration, in relation to the levels in critical normal tissues, is at a maximum. Some boron compounds have recently been proposed for BNCT of malignant melanomas; the synthesized L- and D- forms of carboranylalanine and the previously tested compound L-p-boronophenylalanine are candidates. Human melanoma, IGR1, spheroids were used as models of melanoma nodules in this study. The spheroids developed central necrosis when they were about 480 microns in diameter and the volume doubling time was 2.6 +/- 0.3 days. The tritiated thymidine labelling index decreased rapidly as a function of distance from the periphery and was, at a depth of 175 microns, close to zero. The penetration patterns showed, for L- and D-carboranylalanine and L-p-boronophenylalanine, a homogeneous distribution of 10B throughout the spheroids by 5 min. L-Carboranylalanine gave a more or less even binding of 10B throughout the spheroids and large amounts were present also in the central necrotic regions. D-Carboranylalanine also gave a homogeneous 10B binding in the viable cell layers while the binding in the central necrotic area was lower and a similar, but somewhat lower, binding was found for L-p-boronophenylalanine. Thus, there were no penetration barriers for the boron compounds and binding of 10B was found also in the deeper regions of the viable cell layers. The results showed that the new carboranylalanine compounds are of interest for further analysis, including toxicological and pharmacological studies in vivo.

A facility for biomedical experiments with thermal neutrons.

The Studsvik thermal-neutron facility was originally designed for neutron capture radiography (NCR) at high thermal-neutron fluence and low fast-neutron contamination. It has now been modified to permit irradiation of living cells and animals without the need for stopping and restarting the R2-0 reactor. Previous calculations of the thermal-neutron intensity at the NCR position were performed using the two-dimensional diffusion theory code DIXY. In this report the Monte Carlo program MCNP 3B is used to calculate the neutron fluence at the radiography position, and neutron and photon fluences at the positions of the cell specimens. The normalized neutron fluences from the calculations agree well with measured values and show that the contamination with high-energy neutrons is low. The agreement between measured and calculated photon doses was reasonable. The thermal-neutron fluence rate is (2.3 +/- 0.1) x 10(9) cm-2 s-1 at 100 kW at the NCR position and (0.96 +/- 0.03 x 10(9) cm-2 s-1 at 100 kW at the front plate of the loading tube. The photon dose is about (1.6 +/- 0.2) x 10(-12) Gy per neutron. Survival curves of V79 cells grown as monolayers and irradiated in the thermal neutron beam, with and without boron, are presented.

Quantitative neutron capture radiography for boron in biological specimens.

Track-etch detectors made of cellulose nitrate (LR 115, Kodak Pathé) and polycarbonate (CR 39, Pershore Mouldings Ltd) were compared regarding sensitivity and background when used as detectors for boron determination in biological samples. Measurements were made on two kinds of sample, cryosectioned biological tissue, and liquid samples deposited directly on the detector surface as microdroplets. The CR 39 films were pretreated or washed before irradiation. When cryosectioned tissue was used, measurements were made with and without the inclusion of Mylar foils between the samples and the detectors. Foil thicknesses used were 2 microns in the case of LR 115 and 2, 4, and 6 microns in the case of CR 39. All samples were irradiated with a thermal-neutron fluence of 5 x 10(12) neutrons cm-2 at the thermal-neutron facility in Studsvik, Sweden. The use of a Mylar foil generally suppressed the background tracks relative to the tracks from the 10B disintegration. No difference in resolution between CR 39 and LR 115 was observed. Pretreatment of the CR 39 resulted in an improved sensitivity of detection but the detector became saturated at 0.25 parts per million of 10B. The background was found to be lower in the pretreated detector.

Accumulation of 10B in the central degenerative areas of human glioma and colon carcinoma spheroids after sulfhydryl boron hydride administration.

Sulfhydryl boron hydride (BSH) (10B enriched) is presently used for boron neutron capture therapy of malignant gliomas. BSH must be close to the target cells to be effective in the inactivation of cell proliferation because of the short range of the reaction products (5-9 microns). Clinical experience indicates that BSH is taken up in gliomas but it is not known to which structures it binds at the cellular level. In vitro tests on monolayer cultured cells have indicated that BSH does not bind, or only shows very weak binding, to single isolated cells. It is possible that BSH accumulates in tumor regions due to the special conditions in poorly vascularized tumor tissue, such as low pO2, low extracellular pH, metabolic gradients, and degenerative changes. To test this we incubated three types of multicellular tumor spheroids with BSH for different times and analyzed both penetration and binding. The spatial distribution of 10B in sections of the spheroids was analyzed by neutron capture autoradiography. We found extensive accumulation of 10B in the central regions of both glioma and colon carcinoma spheroids. The accumulation closely followed the pattern of the degenerative changes which were characterized by massive necrosis in the central regions of the colon carcinoma spheroids and by a continuously increasing frequency of pyknotic nuclei as a function of depth in the glioma spheroids. The accumulation of 10B in the prostatic carcinoma spheroids was much lower. The penetration assay, based on freeze-drying and vapor fixation, showed that BSH penetrated easily since 10B equilibrated within 5-15 min in the studied spheroids. Thus, the low accumulation in the prostatic carcinoma spheroids was not due to penetration difficulties. The results of the present study on cellular spheroids and the results from previous studies on transplanted tumors support the observation that BSH penetrates easily into the degenerative tumor areas and that 10B, for some tumor types, might accumulate in these regions as a result of the BSH administration.