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The (11)B(p,alpha)(8)Be* nuclear reaction was assessed for its ability to quantitatively map the in vivo subcellular distribution of boron within gliosarcomas treated with a boronated neutron capture therapy agent. Intracranial 9L gliosarcomas were produced in Fischer 344 rats. Fourteen days later, the majority of the rats were treated with f-boronophenylalanine and killed humanely 30 or 180 min after intravenous injection. Freeze-dried tumor cryosections were imaged using the (11)B(p,alpha)(8)Be* nuclear reaction and proton microbeams obtained from the nuclear microprobe at Lawrence Livermore National Laboratory. The (11)B distributions within cells could be imaged quantitatively with spatial resolutions down to 1.5 microm, minimum detection limits of 0.8 mg/kg, and acquisition times of several hours. These capabilities offer advantages over alpha-particle track autoradiography, electron energy loss spectroscopy, and secondary ion mass spectrometry (SIMS) for quantification of (11)B in tissues. However, the spatial resolution, multi-isotope capability, and analysis times achieved with SIMS are superior to those achieved with (11)B(p,alpha)(8)Be* analysis. When accuracy in quantification is crucial, the (11)B(p,alpha)(8)Be* reaction is well suited for assessing the microdistribution of (11)B. Otherwise, SIMS may well be better suited to image the microdistribution of boron associated with neutron capture therapy agents in biological tissues.

Takagaki, M., Ono, K., Masunaga, S-I., Kinashi, Y., Oda, Y., Miyatake, S-I., Hashimoto, N., Powell, W., Sood, A. and Spielvogel, B. F. Boronated Dipeptide Borotrimethylglycylphenylalanine as a Potential Boron Carrier in Boron Neutron Capture Therapy for Malignant Brain Tumors. Radiat. Res. 156, 118-122 (2001).A boronated dipeptide, borotrimethylglycylphenylalanine (BGPA), was synthesized as a possible boron carrier for boron neutron capture therapy (BNCT) for malignant brain tumors. In vitro, at equal concentrations of (10)B in the extracellular medium, BGPA had the same effect in BNCT as p-boronophenylalanine (BPA). Boron analysis was carried out using prompt gamma-ray spectrometry and track-etch autoradiography. The tumor:blood and tumor:normal brain (10)B concentration ratios were 8.9 +/- 2.1 and 3.0 +/- 1.2, respectively, in rats bearing intracranial C6 gliosarcomas using alpha-particle track autoradiography. The IC(50), i.e. the dose capable of inhibiting the growth of C6 gliosarcoma cells by 50% after 3 days of incubation, was 5.9 x 10(-3) M BGPA, which is similar to that of 6.4 x 10(-3) M for BPA. The amide bond of BGPA is free from enzymatic attack, since it is protected from hydrolysis by the presence of a boron atom at the alpha-carbon position of glycine. These results suggest promise for the use of this agent for BNCT of malignant brain tumors. Further preclinical studies of BGPA are warranted, since BGPA has advantages over both BPA and BSH.

The intracranial 9L tumor model was used to determine if exposure to a radiofrequency (RF) electromagnetic field similar to those used in cellular telephone has any effects on the growth of a central nervous system tumor. Fischer 344 rats implanted with different numbers of 9L gliosarcoma cells were exposed to 835.62 MHz frequency-modulated continuous wave (FMCW) or 847.74 MHz code division multiple access (CDMA) RF field with nominal slot-average specific absorption rates in the brain of 0.75 +/- 0.25 W/kg. The animals were exposed to the RF field for 4 h a day, 5 days a week starting 4 weeks prior to and up to 150 days after the implantation of tumor cells. Among sham-exposed animals injected with 2 to 10 viable cells (group 1), the median survival was 70 days, with 27% of the animals surviving at 150 days. The median survival length and final survival fraction for animals injected with 11 to 36 viable cells (group 2) were 52 days and 14%, respectively, while the values for those injected with 37 to 100 cells (group 3) were 45 days and 0%. The animals exposed to CDMA or FMCW had similar survival parameters, and the statistical comparison of the survival curves for each of the groups 1, 2 and 3 showed no significant differences compared to sham-exposed controls.

The effects of fractionating boron neutron capture therapy (BNCT) were evaluated in the intracerebral rat 9L gliosarcoma and rat spinal cord models using the Brookhaven Medical Research Reactor (BMRR) thermal neutron beam. The amino acid analog p-boronophenylalanine (BPA) was administered prior to each exposure to the thermal neutron beam. The total physical absorbed dose to the tumor during BNCT using BPA was 91% high-linear energy transfer (LET) radiation. Two tumor doses of 5.1 Gy spaced 48 h apart (n = 14) or three tumor doses of 5.2 Gy, each separated by 48 h (n = 10), produced 43 and 70% long-term (1 year) survivors, respectively [corrected]. The outcome of neither the two nor the three fractions of radiation was statistically different from that of the corresponding single-fraction group. In the rat spinal cord, the ED50 for radiation myelopathy (as indicated by limb paralysis within 7 months) after exposure to the thermal beam alone was 13.6 +/- 0.4 Gy. Dividing the beam-only irradiation into two or four consecutive daily fractions increased the ED50 to 14.7 +/- 0.2 Gy and 15.5 +/- 0.4 Gy, respectively. Thermal neutron irradiation in the presence of BPA resulted in an ED50 for myelopathy of 13.8 +/- 0.6 Gy after a single fraction and 14.9 +/- 0.9 Gy after two fractions. An increase in the number of fractions to four resulted in an ED50 of 14.3 +/- 0.6 Gy. The total physical absorbed dose to the blood in the vasculature of the spinal cord during BNCT using BPA was 80% high-LET radiation.(ABSTRACT TRUNCATED AT 250 WORDS)

A well-characterized in vitro cryogenic preparation for ion microscopic isotope imaging, which minimizes redistribution of diffusible species, was used to determine the distribution of boron in GS-9L gliosarcoma cells incubated with the boron neutron capture therapy agent, p-boronophenylalanine (BPA). At the subcellular level, boron from BPA distributes relatively homogeneously within the glioma cell. Boron from BPA was eliminated rapidly, indicating that most is unbound. Thus a large pool of boron is susceptible to diffusion artifact. Removal of this artifact increases the degree of confidence in microdosimetric results inferred from the homogeneous subcellular distribution. The ion microscopic imaging of boron in subcutaneous tumors cryofixed in situ was achieved in rats treated with BPA. Boron signals from BPA were adequate to image microdistributions at the 1-micron resolution level. As in the in vitro case, boron did not localize discretely at the subcellular level. However, boron heterogeneity was seen at the tissue level. Physiologically valid cellular potassium and sodium levels were seen, which demonstrates minimized redistribution artifact. Future tissue studies designed to correlate ion microscopic boron images to microscopic structure are feasible using cryogenic sample preparation and ion microscopy.

The intracranial 9L tumor model was used to determine if exposure to a radiofrequency (RF) electromagnetic field similar to those used in cellular telephone has any effects on the growth of a central nervous system tumor. Fischer 344 rats implanted with different numbers of 9L gliosarcoma cells were exposed to 835.62 MHz frequency-modulated continuous wave (FMCW) or 847.74 MHz code division multiple access (CDMA) RF field with nominal slot-average specific absorption rates in the brain of 0.75 +/- 0.25 W/kg. The animals were exposed to the RF field for 4 h a day, 5 days a week starting 4 weeks prior to and up to 150 days after the implantation of tumor cells. Among sham-exposed animals injected with 2 to 10 viable cells (group 1), the median survival was 70 days, with 27% of the animals surviving at 150 days. The median survival length and final survival fraction for animals injected with 11 to 36 viable cells (group 2) were 52 days and 14%, respectively, while the values for those injected with 37 to 100 cells (group 3) were 45 days and 0%. The animals exposed to CDMA or FMCW had similar survival parameters, and the statistical comparison of the survival curves for each of the groups 1, 2 and 3 showed no significant differences compared to sham-exposed controls.

Photodynamic therapy (PDT) has been used clinically for the treatment of malignant brain gliomas. However, the efficacy of this treatment to date has remained equivocal. This study focused on determining the sensitivity of 9L gliosarcoma in Fischer 344 rats to PDT with increasing doses of 632 nm light and making a comparison of the responses of normal and tumor tissue in the brain at these doses. This sensitivity was then correlated with the concentrations of Photofrin® present in these tissues at the time of treatment. Out study indicates that the level of Photofrin® in the tumor was 13 times that present in normal brain 48 h after injection. However, this selective localization of the photosensitizer was not reflected in a selective tissue response to PDT. There was minimal tumor response to a dose of 35 J cm -2 , which has been reported previously to cause necrosis to the normal brain. Increasing energy dose levels resulted in an increased tumor response to PDT ; however, normal tissue remained more sensitive than tumor tissue at all energy dose levels examined. These data indicate that, although Photofrin® is retained to a significantly higher degree in the tumor than in the normal brain tissue, the normal brain is more sensitive than the tumor to PDT under the conditions outlined in this study.

The effects of fractionating boron neutron capture therapy (BNCT) were evaluated in the intracerebral rat 9L gliosarcoma and rat spinal cord models using the Brookhaven Medical Research Reactor (BMRR) thermal neutron beam. The amino acid analog p-boronophenylalanine (BPA) was administered prior to each exposure to the thermal neutron beam. The total physical absorbed dose to the tumor during BNCT using BPA was 91% high-linear energy transfer (LET) radiation. Two tumor doses of 5.2 Gy spaced 48 h apart (n = 14) or three tumor doses of 5.2 Gy, each separated by 48 h (n = 10), produced 50 and 60% long-term (>1 year) survivors, respectively. The outcome of neither the two nor the three fractions of radiation was statistically different from that of the corresponding single-fraction group. In the rat spinal cord, the ED{sub 50} for radiation myelopathy (as indicated by limb paralysis within 7 months) after exposure to the thermal beam alone was 13.6 {+-} 0.4 Gy. Dividing the beam-only irradiation into two or four consecutive daily fractions increased the ED{sub 50} to 14.7 {+-} 0.2 Gy and 15.5 {+-} 0.4 Gy, respectively. Thermal neutron irradiation in the presence of BPA resulted in an ED{sub 50} for myelopathy ofmore » 13.8 {+-} 0.6 Gy after a single fraction and 14.9 {+-} 0.9 Gy after two fractions. An increase in the number of fractions to four resulted in an ED{sub 50} of 14.3 {+-} 0.6 Gy. The total physical absorbed dose to the blood in the vasculature of the spinal cord during BNCT using BPA was 80% high-LET radiation. It was observed that fractionation was of minor significance in the amelioration of damage to the normal central nervous system in the rat after boron neutron capture irradiation. 30 refs., 5 figs., 3 tabs.« less

A well-characterized in vitro cryogenic preparation for ion microscopic isotope imaging, which minimizes redistribution of diffusible species, was used to determine the distribution of boron in GS-9L gliosarcoma cells incubated with the boron neutron capture therapy agent, p-boronophenylalanine (BPA). At the subcellular level, boron from BPA distributes relatively homogeneously within the glioma cell. Boron from BPA was eliminated rapidly, indicating that most is unbound. Thus a large pool of boron is susceptible to diffusion artifact. Removal of this artifact increases the degree of confidence in microdosimetric results inferred from the homogeneous subcellular distribution. The ion microscopic imaging of boron in subcutaneous tumors cryofixed in situ was achieved in rats treated with BPA. Boron signals from BPA were adequate to image microdistributions at the 1-micron resolution level. As in the in vitro case, boron did not localize discretely at the subcellular level. However, boron heterogeneity was seen at the tissue level. Physiologically valid cellular potassium and sodium levels were seen, which demonstrates minimized redistribution artifact. Future tissue studies designed to correlate ion microscopic boron images to microscopic structure are feasible using cryogenic sample preparation and ion microscopy.

Boron neutron capture therapy (BNCT) of transplanted intracerebral GS-9L rat gliosarcomas was effected by irradiation at a nuclear reactor, primarily with thermal neutrons, after two intragastric doses of p-boronophenylalanine (BPA). At the time of BNCT, tumor10 B levels were ≈40 μg ${}^{10}{\rm B}/{\rm g}$ with tumor:blood and tumor:brain10 B concentration ratios of about 3.3:1 and 3.9:1, respectively. This resulted in calculated doses to tumor that were ≈2.3-fold greater than those to normal brain parenchyma and brain vascular endothelium within the treatment volume. Approximately 75% of the tumor dose resulted from the ${}^{10}{\rm B}({\rm n},\alpha)^{7}{\rm Li}$ nuclear reaction. The median survival of untreated rats (n = 20) was 20 days after initiation of tumors. Reactor irradiation only (no BPA) increased the median survival to 25 days (n = 25). None of the rats in the untreated or irradiation-only groups survived longer than 34 days after initiation of tum...

GUTIN, P. H., HILTON, J., FEIN, V. J., ALLEN, A. E., AND WALKER, M. D. S1 Nuclease from Aspergillus oryzae for the Detection of DNA Damage and Repair in the GammaIrradiated Intracerebral Rat Gliosarcoma 9L. Radiat. Res. 72, 100-106 (1977). DNA damage and repair in a rat brain tumor following irradiation in vivo were measured by analysis of the rate of strand separation of the tumor DNA in alkali. Tumors were removed after irradiation and mechanically dissociated to a cellular suspension. Tumor cells were injected into alkali (pH 12) for 20 min at 220C. The fraction of tumor DNA remaining double-stranded after this exposure to alkali was determined by its resistance to S nuclease from Aspergillus oryzae. Double-stranded DNA remains (after enzyme exposure) acid-precipitable for fluorescent assay. The double-stranded fraction after exposure to alkali decreases with increasing radiation dose following first-order kinetics. DNA from tumors excised at intervals after irradiation showed a greater doublestranded fraction in alkali than that from tumors excised immediately, indicating repair of single-strand breaks. Repair of damage produced by 600 rad proceeded with a half-time of approximately 15 min.

The technique of percentage labeled mitoses was used to compare radiation-induced division delay in 9L rat gliosarcoma cells growing as spheroids or as exponential monolayers. The length of delay induced by each of five X-ray doses was determined as the difference between control and irradiated cultures in the time required to reach the half-height of the first peak of labeled mitoses. Spheroid cells were delayed significantly longer than monolayer cells; the slopes of the dose responses were 32 and 13 min/Gy, respectively. Cells in small spheroids (150 micron diameter) were delayed to the same extent as cells in large spheroids (800 micron diameter). Like the contact effect previously observed as enhanced radiation survival of cells grown as spheroids, the increased radiation-induced delay may be a consequence of the growth of cells in three-dimensional contact.