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First Evaluation of the Biologic Effectiveness Factors of Boron Neutron Capture Therapy (BNCT) in a Human Colon Carcinoma Cell Line
Int. J. Radiation Oncology Biol. Phys., Vol. 79, No. 1, pp. 262–268, 2011 Copyright Ó 2011 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/$–see front matter
doi:10.1016/j.ijrobp.2010.07.020
BIOLOGY CONTRIBUTION
FIRST EVALUATION OF THE BIOLOGIC EFFECTIVENESS FACTORS OF BORON NEUTRON CAPTURE THERAPY (BNCT) IN A HUMAN COLON CARCINOMA CELL LINE MARIA ALEJANDRA DAGROSA, PH.D.,*{ MARTI´N CRIVELLO, M.SC.,* MARINA PERONA, M.SC.,*{ SILVIA THORP, M.SC.,y GUSTAVO ALBERTO SANTA CRUZ, PH.D.,y EMILIANO POZZI, M.SC.,z MARIANA CASAL, M.SC.,x LISA THOMASZ, PH.D.,* ROMULO CABRINI, M.D.,* STEVEN KAHL, PH.D.,** GUILLERMO JUAN JUVENAL, PH.D.,*{ AND MARIO ALBERTO PISAREV, M.D.*{k *Department of Radiobiology and yDepartment of Instrumentation and Control, zArgentina Reactor, National Atomic Energy Commission, Buenos, Argentina; xInstitute of Oncology ‘‘Angel H. Roffo,’’ University of Buenos Aires; {National Research Council; and kDepartment of Human Biochemistry, School of Medicine, University of Buenos Aires, Argentina; and **Department of Pharmaceutical Chemistry, University of California, San Francisco, CA Purpose: DNA lesions produced by boron neutron capture therapy (BNCT) and those produced by gamma radiation in a colon carcinoma cell line were analyzed. We have also derived the relative biologic effectiveness factor (RBE) of the neutron beam of the RA-3- Argentine nuclear reactor, and the compound biologic effectiveness (CBE) values for p-boronophenylalanine (10BPA) and for 2,4-bis (a,b-dihydroxyethyl)-deutero-porphyrin IX (10BOPP). Methods and Materials: Exponentially growing human colon carcinoma cells (ARO81-1) were distributed into the following groups: (1) BPA (10 ppm 10B) + neutrons, (2) BOPP (10 ppm 10B) + neutrons, (3) neutrons alone, and (4) gamma rays (60Co source at 1 Gy/min dose-rate). Different irradiation times were used to obtain total absorbed doses between 0.3 and 5 Gy (±10%) (thermal neutrons flux = 7.5 109 n/cm2 sec). Results: The frequency of micronucleated binucleated cells and the number of micronuclei per micronucleated binucleated cells showed a dose-dependent increase until approximately 2 Gy. The response to gamma rays was significantly lower than the response to the other treatments (p < 0.05). The irradiations with neutrons alone and neutrons + BOPP showed curves that did not differ significantly from, and showed less DNA damage than, irradiation with neutrons + BPA. A decrease in the surviving fraction measured by 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromide (MTT) assay as a function of the absorbed dose was observed for all the treatments. The RBE and CBE factors calculated from cytokinesis block micronucleus (CBMN) and MTT assays were, respectively, the following: beam RBE: 4.4 ± 1.1 and 2.4 ± 0.6; CBE for BOPP: 8.0 ± 2.2 and 2.0 ± 1; CBE for BPA: 19.6 ± 3.7 and 3.5 ± 1.3. Conclusions: BNCT and gamma irradiations showed different genotoxic patterns. To our knowledge, these values represent the first experimental ones obtained for the RA-3 in a biologic model and could be useful for future experimental studies for the application of BNCT to colon carcinoma. Ó 2011 Elsevier Inc. Boron neutron capture therapy, Cancer, Compound biologic effectiveness, Colon, Relative biologic effectiveness.
rounding normal tissues, provided that the boron-carrier compound accumulates preferentially in tumor cells (1). Clinical trials of BNCT are being performed for high-grade gliomas and cutaneous melanomas or their brain metastases using two boron compounds: sodium borocaptate (10BSH) and pboronophenylalanine (10BPA) (2, 3). Although these drugs are safe, they can deliver to the tumor only a limited amount of boron, and therefore other new boron compounds, such as boronated porphyrins, are being analyzed. Tetrakis-
INTRODUCTION Boron neutron capture therapy (BNCT) is a high-linearenergy transfer (LET) radiotherapy for cancer, based on the nuclear reaction that occurs when 10B, a nonradioactive isotope of elemental boron, reacts with low-energy thermal neutrons to produce an alpha particle (4He) and a 7Li nucleus. Both particles have a range comparable to the diameter of a cell causing tumor cell death without significant damage to the sur-
Reprint requests to: M. A. Dagrosa, Ph.D., Department of Radiobiology, CNEA, Av. Del Libertador 8250, Buenos Aires 1429, Argentina. Tel: (54) 11-6772-7966; Fax: (54) 11-6772-7121; E-mail: [email protected] Supported in part by grants from the Fiorini Foundation, National Research Council, and University of Buenos Aires.
Conflict of interest: none. Acknowledgment—The authors thank Dr. Prof. D. Garrido for assistance with the statistical assessment in this work. Received April 7, 2010, and in revised form June 27, 2010. Accepted for publication July 7, 2010. 262
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carborane carboxylate ester of 2,4-bis-(a,b-dihydroxyethyl)deutero-porphyrin IX (BOPP) has been shown to be a good boron carrier in different animal models (4, 5). Recently BNCT has also been applied to the treatment of liver metastases of colon cancer. The Pavia BNCT project developed a method for the treatment of multifocal nonresectable liver metastases of colon carcinoma based on whole liver ex situ BNCT mediated by BPA, followed by whole liver autograft (6). Other biologic experiments of BNCT have been performed, and the efficacy of this therapy has been demonstrated (7). The total absorbed dose in BNCT comes from the contribution of different types of radiation. To express the dose in terms that permit comparison with the conventional treatment (gamma rays or x-rays), each radiation component must be multiplied by the appropriate relative biologic effectiveness (RBE) factor. In the case of the boron component, the compound biologic effectiveness (CBE) factor is used instead of RBE. The CBE factor is specific for different tissues and for different boron compounds and depends largely on the microdistribution of 10B in the cells, which may be influenced by the administration protocol and also by the size and shape of the cells’ nuclei (8, 9). To optimize the dosimetry for the application of BNCT to the treatment of colon carcinoma, it is necessary to estimate the RBE of the beam and the CBE values for this specific tumor and for a particular compound. The DNA molecule is the principal biologic target for radiation-induced damage. Radiation damage in cellular DNA occurs mainly in the form of single- and doublestrand breaks, unrepaired double-strand breaks being generally considered a potentially lethal event. High LET radiation induces double-strand breaks, broken chromosomes, and complex chromosome rearrangements very efficiently, compared to low LET radiation (10). Ionizing radiation can produce cell death by apoptosis or mitotic catastrophe. The latter mode of death is a passive process in which cells pass through mitosis with unrepaired DNA or misrepaired strand breaks, leading to lethal chromosomal aberrations and its cytoplasm derivatives, the micronuclei (MN) in nonclonogenic daughter cells (11). BNCT is a kind of radiotherapy that produces a radiation field of mixed quality in tissues. Therefore, further studies are required to understand the mechanisms that play a role in the tumor damage produced by BNCT. In the present work, we studied the DNA lesions produced by BNCT and compared them with those produced by gamma rays. We also calculated the beam RBE of the RA3 nuclear reactor (Ezeiza Atomic Center, Bs As, Argentina) and the CBE values for BPA and BOPP in a cell line of colon cancer and for two different endpoints: the induction of micronuclei and cell survival. METHODS AND MATERIALS Cell line The cell line ARO81-1 is a subline derived from the colon cancer cell line HT29 provided by Dr. G. Juillard (University of California, Los Angeles, CA). Cells were grown and maintained in RPMI 1640
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medium with glutamine (GIBCO, Invitrogen, Basel, Switzerland) supplemented with 10% of fetal bovine serum, under 5% CO2/ 95% air atmosphere at 37 C. The doubling time was 48 hours (11).
Preparation of boron solutions The stock solution of 10BPA-fructose was prepared at a concentration of 30 mg 10BPA per milliliter (0.14 M). BPA (95% 10B enriched, L-isomer) (New Raleigh, MO) has been described previously (5). The BOPP stock solutions (10 mg/mL) were prepared in phosphate-buffered saline (1) and stored at 4 C in darkness for no more than 24 hours. The experiments with BOPP were performed under conditions of low-intensity light, given that solutions of porphyrins are known to be sensitive to visible light (12).
Experimental design For the micronucleus assay, the ARO81-1 cells were seeded in an amount of 200,000–400,000 and grown in 25-cm2 flasks. For cell survival assay, 2,000 cells/well were seeded in 96-well plates. The exponentially growing cells were irradiated with either thermal neutrons (flux = 7.5 109 n/cm2/sec) or with gamma rays using a 60Co source from the Institute of Oncology Angel H. Roffo (dose rate of 1 Gy/min with an uncertainty of 5%). The cells were distributed into the following groups: (1) BPA + neutrons (BPA + nth): the cells were incubated with BPA at a concentration of 10 ppm of 10B (0.925M) during the 12 hours before irradiation; (2) BOPP + neutrons (BOPP + nth): the cells were incubated with BOPP at a concentration of 10 ppm of 10B (33 mg/mL) during the 16 hours before irradiation; (3) neutrons alone (nth); (4) gamma rays (g). Control groups were performed by incubating the cells with the boron compounds for the same periods of time but without irradiation. The times of incubation were chosen by taking into account previous studies performed on ARO81-1 cells. The boron concentration of the medium in each flask at the end of incubation was checked by inductively coupled plasma optical emission spectroscopy (Perkin-Elmer, Norwalk, CT) as described elsewhere (13).
Dosimetry and characteristics of the neutron irradiation Irradiations were performed in the thermal column irradiation facility of the 8 MW Argentine research nuclear reactor (RA-3). This facility provides a quite uniform thermal neutron flux with negligible contribution from fast neutrons (14). A dosimetric characterization of the irradiation position was previously performed using calibrated rhodium self-powered neutron detectors (Rh-SPND) to determine local thermal flux, which is approximately (7.4 0.6) 109 n/cm2/sec. Gamma dose rate was measured using an air ionization graphite chamber, covered with a LiF shielding cup to avoid neutron interactions in the air. Measurements were performed using the same flasks, at the actual irradiation configuration but with no cells inside. Typical values were near 6.0 0.2 Gy/h, with minor differences for the different kinds of flasks. Total absorbed dose in the cells was estimated by adding partial doses coming from photons, nitrogen capture (a 3.5% weight of nitrogen content was assumed) and boron capture. Nevertheless, before each irradiation, neutron flux at the irradiation position was checked again using a calibrated Rh-SPND at the center of two T25 flasks (with no cells inside), reproducing the configuration that was then used to irradiate the cells while simultaneously a signal from a boron-coated ionization chamber was used as monitor. Based on this measurement, irradiations times were calculated to deliver
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Table 1. Dosimetry for thermal neutron irradiation (nth) without boron (Group 3) Irradiation time (s) 1,135 630 235 80
Thermal neutron flux (n/cm2min1)
Fluence (n/cm2)
Dose g (Gy)
Dose 14N (Gy)
Total dose (Gy)
(4.4 0.4)E11 (4.4 0.4)E11 (4.4 0.4)E11 (4.4 0.4)E11
(8.34 0.05)E12 (4.63 0.03)E12 (1.73 0.01)E12 (5.88 0.05)E11
2.0 0.2 1.1 0.1 0.40 0.05 0.14 0.02
2.3 0.2 1.3 0.1 0.48 0.04 0.16 0.01
4.3 0.3 2.4 0.2 0.89 0.06 0.30 0.02
doses ranging from 0.3 to 5 Gy, with an estimated uncertainty of 10%. Tables 1 and 2 show, for each treatment, the time, the flux, and each dose component of the total absorbed dose of the neutron beam. As there is no reliable method to measure the boron intracellular concentration in vitro at the time of irradiation, we assumed that BPA and BOPP were uniformly distributed inside and outside the cells for the dosimetric calculations. If the boron microdistribution is not uniform, charged particle equilibrium conditions are not fulfilled, and kerma cannot be used as a substitute quantity for absorbed dose. In this case, a microdosimetric calculation must be performed, taking into account the boron accumulation ratio and the average geometric information of the cell-sensitive matrix (8). But since determining the actual boron microdistribution in living cells and their distribution of sizes and shapes is experimentally very difficult and can lead to important uncertainties, we used instead the medium boron concentration for calculating boron kerma, being aware that CBE factors would overestimate or underestimate the boron RBE measured under charged particle equilibrium conditions (e.g., using a compound that distributes uniformly in the medium and in the cells immersed in it).
Cell survival assay At the end of each irradiation assay was performed. After 7 days of incubation at 37 C, the culture medium was changed and 20 mL of 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromide, MTT (Sigma M-2128) 0.5% W/V in phosphate-buffered saline was added to each well. After 4 hours incubation at 37 C, the absorbance was read (540 nm), and the surviving fraction was calculated for each group.
Fitting of the data The data obtained for each studied parameter (MN/BN, % MNBN, and surviving fraction) were plotted as a function of the total absorbed dose (Gy). The dose-surviving fraction data were fitted using a linear quadratic model (ln(S) = aD + bD2), and the dose induction MN values were fitted using a sigmoid function (a/(c+ebD)). From the three obtained curves, the RBE and CBE values were calculated using the following formula (1): RBEðEÞ ¼ Dose gama raysðEÞ=Does beamðEÞ and Dose gama rays ¼ ðDose beamÞ ðRBEÞ þ ðDose--10BÞ ðCBEÞ ðEÞ
Cytokinesis block micronucleus assay For the cytokinesis block micronucleus (CBMN) assay, 4 hours after irradiation, the cell culture medium was removed, and the cells were placed in boron-free culture medium. Cytochalasin B (Sigma, St. Louis, MO), dissolved in dimethyl sulfoxide, was added at a final concentration of 3 mg/mL. After 48 hours, the cells were harvested by trypsinization. rinsed, and submitted to mild hypotonic treatment with 0.075 M KCl for 15 minutes. The centrifuged cells were placed on dry slides. The slides were dried and fixed with cold methanol:acetic acid 3:1 for 30 minutes. After 24 hours, they were stained with 4% Giemsa (Biopur) V/V in H2O for 10 minutes. For each experimental point, 1,000 binucleated (BN) cells with well-preserved cytoplasm were scored. Micronuclei were identified using 500 magnification, according to the criteria described by Caria et al. (15). Two indexes were evaluated: number of micronuclei per binucleated cell (MNBN), representing the average number of MN per BN cell, and frequency of micronucleated binucleated cells (% MNBN), representing the fraction of cytokinesis-blocked BN cells with MN regardless of the number of MN per BN cell.
where E is a given level of effect, assuming that the effects of the different radiation components are additive.
Statistical analysis Data were analyzed by analysis of variance and by Student’s twotailed t test with Welch’s approximation.
RESULTS Figure 1 A and B shows the induction of micronuclei in ARO81-1 cells under the different irradiation treatments. In Fig. 1A the number of MN per binucleated cells (MN/BN) as a function of the total physical dose is shown. The MN/ BN is an important index of the degree of chromosomal lesions per cell. An increase in the MN/BN up to 2 Gy was observed in the cells treated with neutrons and neutrons plus boron (nth; nth + BOPP; nth + BPA). After approximately 2
Table 2. Dosimetry for thermal neutron irradiation (nth plus 10 ppm 10B) (Groups 1 and 2) Irradiation time (s) 420 260 155 52
Thermal neutron flux (n/cm2min1)
Fluence (n/cm2)
Dose g (Gy)
Dose 14N (Gy)
Dose 10B (Gy)
Total dose (Gy)
(4.4 0.4)E11 (4.4 0.4)E11 (4.4 0.4)E11 (4.4 0.4)E11
(3.09 0.02)E12 (1.91 0.01)E12 (1.14 0.01)E12 (3.82 0.04)E11
0.72 0.08 0.45 0.05 0.27 0.03 0.09 0.01
0.86 0.07 0.53 0.04 0.32 0.03 0.11 0.01
2.7 0.6 1.7 0.4 1 0.2 0.33 0.07
4.3 0.6 2.6 0.4 1.6 0.2 0.53 0.08
Biologic effectiveness factors of BNCT in colon carcinoma d M. A. DAGROSA et al.
Fig. 1. (A) Average number of micronuclei per binucleated cell (MN/BN) in ARO81-1 cells under different treatments. Results are expressed as means SEM from two independent experiments. ** p < 0.005 for nth + BOPP; nth + BPA vs g. * p < 0.05 for nth; nth + BOPP; nth + BPA vs g. (B) Frequency of micronucleated binucleated ARO81-1 cells (% MNBN) as a function of the total physical dose. Results are expressed as means SEM from two independent experiments. ** p < 0.005 for nth + BPA vs g. * p < 0.05 for nth; nth + BOPP; nth + BPA vs. g. BOPP = 2,4-bis-(a,b-dihydroxyethyl)deutero-porphyrin; BPA = p-boronophenylalanine.
Gy, saturation was observed. The DNA damage produced by BNCT was significantly greater than the damage produced by gamma rays (p < 0.005). Neutrons alone and neutrons plus BOPP did not show a significant difference. By contrast, BPA was the most effective boron compound, with a maximum average of 2 MN per cell. Figure 1B shows the frequency of micronucleated binucleated cells (% MNBN) as a function of the total absorbed dose. This quantity indicates the level of DNA damage. Saturation in the groups irradiated with neutrons alone or neutrons plus boron was reached with lower doses, indicating that higher doses increase the magnitude of cellular damage but not the proportion of cells with chromosomal lesions. Figure 2 shows the relative frequency of micronucleated binucleated cells for each treatment, classified by the number of MNs that they present (1, 2, 3, 4, and >4) as a function of the absorbed doses. Most of the cells have basically 1 or 2 MN (approximately 50% and 20%, respectively), a pattern
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that is also present in the control ARO81-1 cells (without irradiation). However, in all the cells irradiated with higher doses, a decrease in the percentage of cells with 1 MN and an increase in the percentage of cells with 4 MN or more is observed. A good linear correlation in the cells with 4 or more MNs after all treatments was found, independently of radiation quality. The results for the surviving cell fraction as a function of the total physical dose are shown in Figure 3. Neutron irradiation produced a decrease in cell viability. As observed for the induction of micronuclei, this effect was also greater in the cells incubated with BPA. Neutrons alone and neutrons plus BOPP did not show significant differences. The beam RBE and the CBE factors for BPA and BOPP were calculated from the data shown in Fig. 1A and B and Fig. 3 using the expressions described above. The experimental values obtained are shown in Table 3. The beam RBE values varied from 2.6 0.7 to 4.4 1.1 for the CBMN assay. The surviving fraction (MTT) as endpoint gave values of 2.3 0.6 and 2.4 0.6. The derived calculated CBE values for BPA and BOPP were higher for the CBMN assay than for the MTT assay. The obtained values for BPA were between 12.2 2.3 and 19.6 3.7 for the DNA damage and 3.5 1.2 and 3.7 1.3 for surviving fraction, respectively. By contrast, the CBE factors calculated for BOPP were 2.9 1.1 and 8.0 2.2 when the endpoint was DNA damage and 1.9 1.0 and 2.0 1.0 when the endpoint was the surviving fraction. These results confirmed that BPA was the most effective compound for the application of BNCT to the treatment of colon carcinoma. DISCUSSION We studied the induction of micronuclei in binucleated cells (CBMN) in a cell line of colon carcinoma. Micronuclei induction is an indicator of chromosomal damage caused directly by radiation or by DNA damage misrepair. In our studies the response to neutrons plus BOPP was similar to the response to treatment with the neutron beam alone for measured biologic effects, DNA damage, and surviving fraction. This finding can be explained if boron cell localization is taken into account. The porphyrin is more concentrated outside the cells, and the fraction that enters the cell is localized in the mitochondrial and lysosomal fractions (5). Monte Carlo simulations showed that for cells 10 mm in diameter when boron is localized outside the cell membrane the physical dose delivered to the nuclei is only 10% of that obtained when boron is distributed uniformly in the entire cell (16). Considering this information, the boron kerma calculated using the medium concentration is likely to be an overestimation of the absorbed boron dose to the cell nuclei, explaining the seemingly low effectiveness of BOPP. The CBE factors obtained therefore underestimate the actual boron RBEs (8). However, CBE factors remain the only meaningful option for calculating biologically weighted doses, because they intrinsically contain the effect of the microscopic nonuniformity. In this sense, they are different from
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Fig. 2. Distribution of the number of micronuclei (MN) per ARO81-1 binucleated cell in the different treatments. (A) Gamma radiation. (B) Thermal neutrons. (C) Neutrons + BOPP. (D) Neutrons + BPA. Average number of two independent experiments. BOPP = 2,4-bis-(a,b-dihydroxyethyl)-deutero-porphyrin; BPA = p-boronophenylalanine.
a pure RBE because they include the influence of cell morphology and boron average microdistribution on the actual dose delivered to cells.
Fig. 3. Survival of ARO81-1 cells under the different treatments. Data are fitted with a linear quadratic model. Each point is the average of six to eight wells SEM of two independent experiments. BOPP = 2,4-bis-(a,b-dihydroxyethyl)-deutero-porphyrin; BPA = p-boronophenylalanine.
In our studies we observed a small percentage of spontaneous DNA damage in control cells for the different treatments (no irradiation), which has also been reported in other tumor cell lines but not observed in normal cells (17). On the other hand, we can exclude that BPA and BOPP have genotoxic effects per se in the administered doses. These results agree with studies published elsewhere that have shown no cytotoxic or genotoxic effects for these boron compounds (18, 19). The dose–response curves obtained for the induction of micronuclei showed that both types of radiation (high and low LET) can be fitted with a sigmoid function. One explanation for this behavior may be that at low doses (approximately 2 Gy) the number of chromosome fragments can be low enough for them to have a high possibility of being expressed as a single MN each. At higher doses, the increased number of fragments can lead to two or more being incorporated into a single MN, thus explaining the leveling off in the yield of micronuclei. Furthermore, other authors attribute the depression in the induction of MN, as a function of dose, to the apoptotic process in an important number of cells (20). By contrast, our results show that the curves for low LET radiation (MN/BN; MNBN) can also be fitted with a linear function (R = 0.96) in agreement with other published results (21).
Biologic effectiveness factors of BNCT in colon carcinoma d M. A. DAGROSA et al.
Table 3. Experimental RBE and CBEs for colon carcinoma Parameter MN/BN % MN/BN Surviving fraction (MTT)
Endpoint level
Beam RBE
CBE BOPP
CBE BPA
0.8 1.2 43.4 52.0 65.6 75.8
2.6 0.7 4.2 1.1 2.6 0.7 4.4 1.1 2.4 0.6 2.3 0.6
4.6 1.1 8.0 2.2 2.9 1.1 5.1 1.9 2.0 1.0 1.9 1.0
12.2 2.3 19.6 3.7 12.3 2.3 17.8 3.6 3.5 1.2 3.7 1.3
Abbreviations: RBE = relative biologic effectiveness; CBE = compound biologic effectiveness; BOPP = 2,4-bis-(a,b-dihydroxyethyl)-deutero-porphyrin IX; BPA = p-boronophenylalanine; MN = micronuclei; BN = binucleated; MTT = 3-(4,5-dimetiltiazol-2il)-2,5-difeniltetrazolium bromide assay. Experimental RBE and CBE for colon carcinoma were calculated. In vitro cells were subject to four treatments. Two endpoints were measured: micronuclei induction and surviving fraction.
The reliability of the MNBN assay is controversial (22). It is known that reproductive cell death does not occur only before cell division, leading to overestimating the amount of cells that reach mitotic death because of the treatment. Also in some conditions (mostly for high doses of radiation), it can be difficult to distinguish between cells with only a nucleus from binucleated cells that have lost a nucleus after rupture caused by the manipulation of the samples, leading then to an underestimation of the actual number of binucleated cells. However, in our studies a good correlation between the induction of MN and the surviving fraction was found (R = 0.95, result not shown). As mentioned before, the calculated relative biologic effectiveness factors of the neutron beam (RBE) and of the boron compound (CBE) are required for estimating the biologically weighted dose delivered in BNCT, in our case for experimental colon carcinoma models. Each factor multiplies each of the components of the absorbed doses, thus allowing dose values to be expressed as biologically weighted dose. As it is known, these correction factors vary according to tissue composition and its specific radiosensitivity. In addition, RBE factors vary depending on the gamma and neutron spectra as a function of depth in tissue and dose levels,
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thus being different for each BNCT facility (23). Despite all the variables considered, the values obtained in this work are in the same range of those obtained by other authors in other tumor tissues and reactors (24, 25). Therefore, it is not surprising that RBE values for micronuclei induction are much higher than for cell inactivation when the values found elsewhere are taken into account, considering, though, that the cell line analyzed in this work is of tumoral origin and that RBE values depend strongly on the cell type. However, investigating the dose dependence of RBE rather than effect probability has major advantages, inasmuch as it is believed that features such as lesion formation, repair kinetics, and the assay chosen are factors that cancel out when taking the ratio of doses that produce the same level of effect (26). In fact, this work shows that the RBE hierarchy expected between chromosome aberration formation and cell killing is preserved. Special consideration must be taken in terms of transferring RBE values obtained from in vitro experiments to the in vivo situation, although this issue is of general concern in BNCT. Although tumor cells lack the typical high-level organization of normal tissues and so their response to radiation can be associated more easily with a parallel-organized tissue, which permits a more direct association with cell culture experiments, the main factor that obscures the direct transfer is the actual boron uptake in tumor cells. Several barriers must be crossed by a compound from the bloodstream until it reaches the target cell population (27). Colon carcinoma is the third most frequent malignant neoplasm in Western countries. After a complete resection, 5-year overall survival varies according to the stage at diagnosis (28). The development of new therapies is necessary. BNCT was applied for the treatment of liver metastases of colon carcinoma in a clinical setting at Pavia (6). Other groups started biodistribution studies of BPA and BSH in human patients with liver metastases of the same carcinoma (29, 30). The values obtained in these studies, to our knowledge, would be the first ones published for a model of experimental colon carcinoma in BNCT. It is also worth mentioning that these factors are the first experimental and biologic values obtained in the RA-3 facility.
REFERENCES 1. Coderre JA, Morris GM. The radiation biology of boron neutron capture therapy. Radiat Res 1999;1:1–18. 2. Busse PM, Harling OK, Palmer MR, et al. A critical examination of the results from the Harvard-MIT NCT program phase I clinical trial of neutron capture therapy for intracranial disease. J Neurooncol 2003;62:111–121. 3. Hill J, Kahl SB, Kaye A, et al. Selective tumor uptake of a boronated porphyrin in an animal model of cerebral glioma. Proc Natl Acad Sci U S A 1992;89:1785–1789. 4. Dagrosa MA, Viaggi ME, Jime´nez Rebagliati R, et al. Biodistribution of boron compounds in an animal model of undifferentiated thyroid cancer for boron neutron capture therapy. Mol Pharm 2005;2:151–156. 5. Pinelli T, Zonta A, Altieri S, et al. Taormina: From the first idea to the application to the human liver. In: Sauerwein W, Moss R,
6.
7. 8. 9.
Wittig A, editors. Research and development in neutron capture therapy. Bologna, Italy: Monduzzi Editore; 2002. p. 1065– 1072. Zonta A, Pinelli T, Prati U, et al. Extra-corporeal liver BNCT for the treatment of diffuse metastases: What was learned and what is still to be learned. Appl Radiat Isot 2009;67:S67– S75. Coderre J, Turcotte J, Riley K, et al. Boron neutron capture therapy: Cellular targeting of high linear energy transfer radiation. Technol Cancer Res Treat 2003;2:355. Santa Cruz GA, Zamenhof RG. The microdosimetry of the 10B reaction in boron neutron capture therapy: A new generalized theory. Radiat Res 2004;162:702–710. Fox J, Prise K. DNA lesions: Linear energy transfer and radiosensitive mutants. BJR Suppl 1992;24:48–52.
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10. Revell S. Radiation induced chromosome damage in man. In: Ishihara T, Sasaki M, editors. Relationship between chromosome damage and cell death. New York: Liss; 1983. p. 215–233. 11. Schweppe RE, Klopper JP, Korch C, et al. Deoxyribonucleic and profiling analysis of 40 human thyroid cancer cell lines reveals cross-contamination resulting in cell line redundancy and misidentification. J Clin Endocrinol Metab 2008;93:4331– 4341. 12. Kahl SB, Koo MS. Synthesis and properties of tetrakiscarboranecarboxylate esters of 2, 4-bis (-dihydroxyethyl) deuteroporphyrin IX. In: Allen BJ, Moore DE, Harrington BV, editors. Progress in neutron capture therapy for cancer. New York: Plenum Press; 1992. p. 223–226. 13. Dagrosa MA, Viaggi ME, Kreimann EL, et al. Selective uptake of p-borophenylalanine by undifferentiated thyroid carcinoma for boron neutron capture therapy. Thyroid 2002;12:7–12. 14. Miller M, Quintana J, Ojeda J, et al. New irradiation facility for biomedical applications at the RA-3 reactor thermal column. Appl Radiat Isotopes 2009;67:226–229. 15. Caria H, Chaveca T, Laires A, et al. Genotoxicity of quercentin in the micronucleus assay in mouse bone marrow erythrocytes, human lymphocytes, V79 cell line and identification of kinetochore-containing (CREST staining) micronuclei in human lymphocytes. Mutat Res 1995;343:85–94. 16. Gabel D, Foster S, Fairchild RG. The Monte Carlo simulation of the biological effect of the 10B (n,a) 7Li reaction in cells and tissues and its implication for boron neutron capture therapy. Radiat Res 1987;111:14–25. 17. Champion A, Hanson J, Court J, et al. The micronucleus assay: An evaluation of its use in determining radiosensitivity in vitro. Mutagenesis 1995;10:203–208. 18. Oliveira N, Castro M, Rodriguez A, et al. Evaluation of the genotoxic effect of the boron neutron capture reaction in human melanoma cells using the cytokinesis block micronucleus assay. Mutagenesis 2001;16:369–375. 19. Kinashi Y, Masunaga S, Ono K. Mutagenic effect of borocaptate sodium and boronophenylalanine in neutron cap-
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20. 21. 22. 23.
24.
25. 26. 27. 28. 29.
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ture therapy. Int J Radiat Oncol Biol Phys 2002;51:562– 567. Belyakov O, Prise K, Trott K, et al. Delayed lethality, apoptosis and micronucleus formation in human fibroblasts irradiated with X-rays or alpha-particles. Int J Radiat Biol 1999;75:985–993. Bedford J, Goodhead D. Breakage of human interphase chromosomes by alpha particles and X rays. Int J Radiat Biol 1989;55:211–216. Savage JRK. Micronuclei: Pitfalls and problems. Atlas Genet Cytogenet Oncol Haematol July 2000. Available at: http:// AtlasGenetics Oncology.org/Deep/MicronucleiID20016.html Gueulette J, Octave-Prignot M, De Costera B, et al. Intestinal crypt regeneration in mice: A biological system for quality assurance in non-conventional radiation therapy. Radiother Oncol 2004;73:148–154. Coderre J, Makar M, Micca P, et al. Derivations of relative biological effectiveness for the high-LET radiations produced during boron neutron capture irradiation of the 9L rat gliosarcoma in vitro and. in vivo. Int J Radiat Oncol Biol Phys 1993; 27:1121–1129. Suzuki M, Masunaga S, Kinashi Y, et al. The effects of boron neutron capture therapy on liver tumors and normal hepatocytes in mice. Jpn J Cancer Res 2000;91:1058–1064. Rossi HH, Zaider M. Microdosimetry and its applications. Berlin, Heidelberg, New York: Springer; 1996. p. 230. Jain R. Interstitial transport in tumors: Barriers and strategies for improvement. AACR 96th Annual Meeting 2005;108–113. Gravalos C, Garcia-Escobar I, Garcia-Alfonso P, et al. Adjuvant chemotherapy for stages II, III and IV of colon cancer. Clin Transl Oncol 2009;11:526–533. Wittig A, Malago M, Collete L, et al. Uptake of two 10B compounds in liver metastases of colorectal adenocarcinoma for extracorporeal irradiation with boron neutron capture therapy (EORTC Trial 11001). Int J Cancer 2008;122:1164–1171. Cardoso J, Nievas S, Pereira D, et al. Boron biodistribution study in colorectal liver metastases patients in Argentina. Appl Radiat Isot 2009;67:576–579.
doi:10.1016/j.ijrobp.2010.07.020
BIOLOGY CONTRIBUTION
FIRST EVALUATION OF THE BIOLOGIC EFFECTIVENESS FACTORS OF BORON NEUTRON CAPTURE THERAPY (BNCT) IN A HUMAN COLON CARCINOMA CELL LINE MARIA ALEJANDRA DAGROSA, PH.D.,*{ MARTI´N CRIVELLO, M.SC.,* MARINA PERONA, M.SC.,*{ SILVIA THORP, M.SC.,y GUSTAVO ALBERTO SANTA CRUZ, PH.D.,y EMILIANO POZZI, M.SC.,z MARIANA CASAL, M.SC.,x LISA THOMASZ, PH.D.,* ROMULO CABRINI, M.D.,* STEVEN KAHL, PH.D.,** GUILLERMO JUAN JUVENAL, PH.D.,*{ AND MARIO ALBERTO PISAREV, M.D.*{k *Department of Radiobiology and yDepartment of Instrumentation and Control, zArgentina Reactor, National Atomic Energy Commission, Buenos, Argentina; xInstitute of Oncology ‘‘Angel H. Roffo,’’ University of Buenos Aires; {National Research Council; and kDepartment of Human Biochemistry, School of Medicine, University of Buenos Aires, Argentina; and **Department of Pharmaceutical Chemistry, University of California, San Francisco, CA Purpose: DNA lesions produced by boron neutron capture therapy (BNCT) and those produced by gamma radiation in a colon carcinoma cell line were analyzed. We have also derived the relative biologic effectiveness factor (RBE) of the neutron beam of the RA-3- Argentine nuclear reactor, and the compound biologic effectiveness (CBE) values for p-boronophenylalanine (10BPA) and for 2,4-bis (a,b-dihydroxyethyl)-deutero-porphyrin IX (10BOPP). Methods and Materials: Exponentially growing human colon carcinoma cells (ARO81-1) were distributed into the following groups: (1) BPA (10 ppm 10B) + neutrons, (2) BOPP (10 ppm 10B) + neutrons, (3) neutrons alone, and (4) gamma rays (60Co source at 1 Gy/min dose-rate). Different irradiation times were used to obtain total absorbed doses between 0.3 and 5 Gy (±10%) (thermal neutrons flux = 7.5 109 n/cm2 sec). Results: The frequency of micronucleated binucleated cells and the number of micronuclei per micronucleated binucleated cells showed a dose-dependent increase until approximately 2 Gy. The response to gamma rays was significantly lower than the response to the other treatments (p < 0.05). The irradiations with neutrons alone and neutrons + BOPP showed curves that did not differ significantly from, and showed less DNA damage than, irradiation with neutrons + BPA. A decrease in the surviving fraction measured by 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromide (MTT) assay as a function of the absorbed dose was observed for all the treatments. The RBE and CBE factors calculated from cytokinesis block micronucleus (CBMN) and MTT assays were, respectively, the following: beam RBE: 4.4 ± 1.1 and 2.4 ± 0.6; CBE for BOPP: 8.0 ± 2.2 and 2.0 ± 1; CBE for BPA: 19.6 ± 3.7 and 3.5 ± 1.3. Conclusions: BNCT and gamma irradiations showed different genotoxic patterns. To our knowledge, these values represent the first experimental ones obtained for the RA-3 in a biologic model and could be useful for future experimental studies for the application of BNCT to colon carcinoma. Ó 2011 Elsevier Inc. Boron neutron capture therapy, Cancer, Compound biologic effectiveness, Colon, Relative biologic effectiveness.
rounding normal tissues, provided that the boron-carrier compound accumulates preferentially in tumor cells (1). Clinical trials of BNCT are being performed for high-grade gliomas and cutaneous melanomas or their brain metastases using two boron compounds: sodium borocaptate (10BSH) and pboronophenylalanine (10BPA) (2, 3). Although these drugs are safe, they can deliver to the tumor only a limited amount of boron, and therefore other new boron compounds, such as boronated porphyrins, are being analyzed. Tetrakis-
INTRODUCTION Boron neutron capture therapy (BNCT) is a high-linearenergy transfer (LET) radiotherapy for cancer, based on the nuclear reaction that occurs when 10B, a nonradioactive isotope of elemental boron, reacts with low-energy thermal neutrons to produce an alpha particle (4He) and a 7Li nucleus. Both particles have a range comparable to the diameter of a cell causing tumor cell death without significant damage to the sur-
Reprint requests to: M. A. Dagrosa, Ph.D., Department of Radiobiology, CNEA, Av. Del Libertador 8250, Buenos Aires 1429, Argentina. Tel: (54) 11-6772-7966; Fax: (54) 11-6772-7121; E-mail: [email protected] Supported in part by grants from the Fiorini Foundation, National Research Council, and University of Buenos Aires.
Conflict of interest: none. Acknowledgment—The authors thank Dr. Prof. D. Garrido for assistance with the statistical assessment in this work. Received April 7, 2010, and in revised form June 27, 2010. Accepted for publication July 7, 2010. 262
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carborane carboxylate ester of 2,4-bis-(a,b-dihydroxyethyl)deutero-porphyrin IX (BOPP) has been shown to be a good boron carrier in different animal models (4, 5). Recently BNCT has also been applied to the treatment of liver metastases of colon cancer. The Pavia BNCT project developed a method for the treatment of multifocal nonresectable liver metastases of colon carcinoma based on whole liver ex situ BNCT mediated by BPA, followed by whole liver autograft (6). Other biologic experiments of BNCT have been performed, and the efficacy of this therapy has been demonstrated (7). The total absorbed dose in BNCT comes from the contribution of different types of radiation. To express the dose in terms that permit comparison with the conventional treatment (gamma rays or x-rays), each radiation component must be multiplied by the appropriate relative biologic effectiveness (RBE) factor. In the case of the boron component, the compound biologic effectiveness (CBE) factor is used instead of RBE. The CBE factor is specific for different tissues and for different boron compounds and depends largely on the microdistribution of 10B in the cells, which may be influenced by the administration protocol and also by the size and shape of the cells’ nuclei (8, 9). To optimize the dosimetry for the application of BNCT to the treatment of colon carcinoma, it is necessary to estimate the RBE of the beam and the CBE values for this specific tumor and for a particular compound. The DNA molecule is the principal biologic target for radiation-induced damage. Radiation damage in cellular DNA occurs mainly in the form of single- and doublestrand breaks, unrepaired double-strand breaks being generally considered a potentially lethal event. High LET radiation induces double-strand breaks, broken chromosomes, and complex chromosome rearrangements very efficiently, compared to low LET radiation (10). Ionizing radiation can produce cell death by apoptosis or mitotic catastrophe. The latter mode of death is a passive process in which cells pass through mitosis with unrepaired DNA or misrepaired strand breaks, leading to lethal chromosomal aberrations and its cytoplasm derivatives, the micronuclei (MN) in nonclonogenic daughter cells (11). BNCT is a kind of radiotherapy that produces a radiation field of mixed quality in tissues. Therefore, further studies are required to understand the mechanisms that play a role in the tumor damage produced by BNCT. In the present work, we studied the DNA lesions produced by BNCT and compared them with those produced by gamma rays. We also calculated the beam RBE of the RA3 nuclear reactor (Ezeiza Atomic Center, Bs As, Argentina) and the CBE values for BPA and BOPP in a cell line of colon cancer and for two different endpoints: the induction of micronuclei and cell survival. METHODS AND MATERIALS Cell line The cell line ARO81-1 is a subline derived from the colon cancer cell line HT29 provided by Dr. G. Juillard (University of California, Los Angeles, CA). Cells were grown and maintained in RPMI 1640
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medium with glutamine (GIBCO, Invitrogen, Basel, Switzerland) supplemented with 10% of fetal bovine serum, under 5% CO2/ 95% air atmosphere at 37 C. The doubling time was 48 hours (11).
Preparation of boron solutions The stock solution of 10BPA-fructose was prepared at a concentration of 30 mg 10BPA per milliliter (0.14 M). BPA (95% 10B enriched, L-isomer) (New Raleigh, MO) has been described previously (5). The BOPP stock solutions (10 mg/mL) were prepared in phosphate-buffered saline (1) and stored at 4 C in darkness for no more than 24 hours. The experiments with BOPP were performed under conditions of low-intensity light, given that solutions of porphyrins are known to be sensitive to visible light (12).
Experimental design For the micronucleus assay, the ARO81-1 cells were seeded in an amount of 200,000–400,000 and grown in 25-cm2 flasks. For cell survival assay, 2,000 cells/well were seeded in 96-well plates. The exponentially growing cells were irradiated with either thermal neutrons (flux = 7.5 109 n/cm2/sec) or with gamma rays using a 60Co source from the Institute of Oncology Angel H. Roffo (dose rate of 1 Gy/min with an uncertainty of 5%). The cells were distributed into the following groups: (1) BPA + neutrons (BPA + nth): the cells were incubated with BPA at a concentration of 10 ppm of 10B (0.925M) during the 12 hours before irradiation; (2) BOPP + neutrons (BOPP + nth): the cells were incubated with BOPP at a concentration of 10 ppm of 10B (33 mg/mL) during the 16 hours before irradiation; (3) neutrons alone (nth); (4) gamma rays (g). Control groups were performed by incubating the cells with the boron compounds for the same periods of time but without irradiation. The times of incubation were chosen by taking into account previous studies performed on ARO81-1 cells. The boron concentration of the medium in each flask at the end of incubation was checked by inductively coupled plasma optical emission spectroscopy (Perkin-Elmer, Norwalk, CT) as described elsewhere (13).
Dosimetry and characteristics of the neutron irradiation Irradiations were performed in the thermal column irradiation facility of the 8 MW Argentine research nuclear reactor (RA-3). This facility provides a quite uniform thermal neutron flux with negligible contribution from fast neutrons (14). A dosimetric characterization of the irradiation position was previously performed using calibrated rhodium self-powered neutron detectors (Rh-SPND) to determine local thermal flux, which is approximately (7.4 0.6) 109 n/cm2/sec. Gamma dose rate was measured using an air ionization graphite chamber, covered with a LiF shielding cup to avoid neutron interactions in the air. Measurements were performed using the same flasks, at the actual irradiation configuration but with no cells inside. Typical values were near 6.0 0.2 Gy/h, with minor differences for the different kinds of flasks. Total absorbed dose in the cells was estimated by adding partial doses coming from photons, nitrogen capture (a 3.5% weight of nitrogen content was assumed) and boron capture. Nevertheless, before each irradiation, neutron flux at the irradiation position was checked again using a calibrated Rh-SPND at the center of two T25 flasks (with no cells inside), reproducing the configuration that was then used to irradiate the cells while simultaneously a signal from a boron-coated ionization chamber was used as monitor. Based on this measurement, irradiations times were calculated to deliver
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Table 1. Dosimetry for thermal neutron irradiation (nth) without boron (Group 3) Irradiation time (s) 1,135 630 235 80
Thermal neutron flux (n/cm2min1)
Fluence (n/cm2)
Dose g (Gy)
Dose 14N (Gy)
Total dose (Gy)
(4.4 0.4)E11 (4.4 0.4)E11 (4.4 0.4)E11 (4.4 0.4)E11
(8.34 0.05)E12 (4.63 0.03)E12 (1.73 0.01)E12 (5.88 0.05)E11
2.0 0.2 1.1 0.1 0.40 0.05 0.14 0.02
2.3 0.2 1.3 0.1 0.48 0.04 0.16 0.01
4.3 0.3 2.4 0.2 0.89 0.06 0.30 0.02
doses ranging from 0.3 to 5 Gy, with an estimated uncertainty of 10%. Tables 1 and 2 show, for each treatment, the time, the flux, and each dose component of the total absorbed dose of the neutron beam. As there is no reliable method to measure the boron intracellular concentration in vitro at the time of irradiation, we assumed that BPA and BOPP were uniformly distributed inside and outside the cells for the dosimetric calculations. If the boron microdistribution is not uniform, charged particle equilibrium conditions are not fulfilled, and kerma cannot be used as a substitute quantity for absorbed dose. In this case, a microdosimetric calculation must be performed, taking into account the boron accumulation ratio and the average geometric information of the cell-sensitive matrix (8). But since determining the actual boron microdistribution in living cells and their distribution of sizes and shapes is experimentally very difficult and can lead to important uncertainties, we used instead the medium boron concentration for calculating boron kerma, being aware that CBE factors would overestimate or underestimate the boron RBE measured under charged particle equilibrium conditions (e.g., using a compound that distributes uniformly in the medium and in the cells immersed in it).
Cell survival assay At the end of each irradiation assay was performed. After 7 days of incubation at 37 C, the culture medium was changed and 20 mL of 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromide, MTT (Sigma M-2128) 0.5% W/V in phosphate-buffered saline was added to each well. After 4 hours incubation at 37 C, the absorbance was read (540 nm), and the surviving fraction was calculated for each group.
Fitting of the data The data obtained for each studied parameter (MN/BN, % MNBN, and surviving fraction) were plotted as a function of the total absorbed dose (Gy). The dose-surviving fraction data were fitted using a linear quadratic model (ln(S) = aD + bD2), and the dose induction MN values were fitted using a sigmoid function (a/(c+ebD)). From the three obtained curves, the RBE and CBE values were calculated using the following formula (1): RBEðEÞ ¼ Dose gama raysðEÞ=Does beamðEÞ and Dose gama rays ¼ ðDose beamÞ ðRBEÞ þ ðDose--10BÞ ðCBEÞ ðEÞ
Cytokinesis block micronucleus assay For the cytokinesis block micronucleus (CBMN) assay, 4 hours after irradiation, the cell culture medium was removed, and the cells were placed in boron-free culture medium. Cytochalasin B (Sigma, St. Louis, MO), dissolved in dimethyl sulfoxide, was added at a final concentration of 3 mg/mL. After 48 hours, the cells were harvested by trypsinization. rinsed, and submitted to mild hypotonic treatment with 0.075 M KCl for 15 minutes. The centrifuged cells were placed on dry slides. The slides were dried and fixed with cold methanol:acetic acid 3:1 for 30 minutes. After 24 hours, they were stained with 4% Giemsa (Biopur) V/V in H2O for 10 minutes. For each experimental point, 1,000 binucleated (BN) cells with well-preserved cytoplasm were scored. Micronuclei were identified using 500 magnification, according to the criteria described by Caria et al. (15). Two indexes were evaluated: number of micronuclei per binucleated cell (MNBN), representing the average number of MN per BN cell, and frequency of micronucleated binucleated cells (% MNBN), representing the fraction of cytokinesis-blocked BN cells with MN regardless of the number of MN per BN cell.
where E is a given level of effect, assuming that the effects of the different radiation components are additive.
Statistical analysis Data were analyzed by analysis of variance and by Student’s twotailed t test with Welch’s approximation.
RESULTS Figure 1 A and B shows the induction of micronuclei in ARO81-1 cells under the different irradiation treatments. In Fig. 1A the number of MN per binucleated cells (MN/BN) as a function of the total physical dose is shown. The MN/ BN is an important index of the degree of chromosomal lesions per cell. An increase in the MN/BN up to 2 Gy was observed in the cells treated with neutrons and neutrons plus boron (nth; nth + BOPP; nth + BPA). After approximately 2
Table 2. Dosimetry for thermal neutron irradiation (nth plus 10 ppm 10B) (Groups 1 and 2) Irradiation time (s) 420 260 155 52
Thermal neutron flux (n/cm2min1)
Fluence (n/cm2)
Dose g (Gy)
Dose 14N (Gy)
Dose 10B (Gy)
Total dose (Gy)
(4.4 0.4)E11 (4.4 0.4)E11 (4.4 0.4)E11 (4.4 0.4)E11
(3.09 0.02)E12 (1.91 0.01)E12 (1.14 0.01)E12 (3.82 0.04)E11
0.72 0.08 0.45 0.05 0.27 0.03 0.09 0.01
0.86 0.07 0.53 0.04 0.32 0.03 0.11 0.01
2.7 0.6 1.7 0.4 1 0.2 0.33 0.07
4.3 0.6 2.6 0.4 1.6 0.2 0.53 0.08
Biologic effectiveness factors of BNCT in colon carcinoma d M. A. DAGROSA et al.
Fig. 1. (A) Average number of micronuclei per binucleated cell (MN/BN) in ARO81-1 cells under different treatments. Results are expressed as means SEM from two independent experiments. ** p < 0.005 for nth + BOPP; nth + BPA vs g. * p < 0.05 for nth; nth + BOPP; nth + BPA vs g. (B) Frequency of micronucleated binucleated ARO81-1 cells (% MNBN) as a function of the total physical dose. Results are expressed as means SEM from two independent experiments. ** p < 0.005 for nth + BPA vs g. * p < 0.05 for nth; nth + BOPP; nth + BPA vs. g. BOPP = 2,4-bis-(a,b-dihydroxyethyl)deutero-porphyrin; BPA = p-boronophenylalanine.
Gy, saturation was observed. The DNA damage produced by BNCT was significantly greater than the damage produced by gamma rays (p < 0.005). Neutrons alone and neutrons plus BOPP did not show a significant difference. By contrast, BPA was the most effective boron compound, with a maximum average of 2 MN per cell. Figure 1B shows the frequency of micronucleated binucleated cells (% MNBN) as a function of the total absorbed dose. This quantity indicates the level of DNA damage. Saturation in the groups irradiated with neutrons alone or neutrons plus boron was reached with lower doses, indicating that higher doses increase the magnitude of cellular damage but not the proportion of cells with chromosomal lesions. Figure 2 shows the relative frequency of micronucleated binucleated cells for each treatment, classified by the number of MNs that they present (1, 2, 3, 4, and >4) as a function of the absorbed doses. Most of the cells have basically 1 or 2 MN (approximately 50% and 20%, respectively), a pattern
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that is also present in the control ARO81-1 cells (without irradiation). However, in all the cells irradiated with higher doses, a decrease in the percentage of cells with 1 MN and an increase in the percentage of cells with 4 MN or more is observed. A good linear correlation in the cells with 4 or more MNs after all treatments was found, independently of radiation quality. The results for the surviving cell fraction as a function of the total physical dose are shown in Figure 3. Neutron irradiation produced a decrease in cell viability. As observed for the induction of micronuclei, this effect was also greater in the cells incubated with BPA. Neutrons alone and neutrons plus BOPP did not show significant differences. The beam RBE and the CBE factors for BPA and BOPP were calculated from the data shown in Fig. 1A and B and Fig. 3 using the expressions described above. The experimental values obtained are shown in Table 3. The beam RBE values varied from 2.6 0.7 to 4.4 1.1 for the CBMN assay. The surviving fraction (MTT) as endpoint gave values of 2.3 0.6 and 2.4 0.6. The derived calculated CBE values for BPA and BOPP were higher for the CBMN assay than for the MTT assay. The obtained values for BPA were between 12.2 2.3 and 19.6 3.7 for the DNA damage and 3.5 1.2 and 3.7 1.3 for surviving fraction, respectively. By contrast, the CBE factors calculated for BOPP were 2.9 1.1 and 8.0 2.2 when the endpoint was DNA damage and 1.9 1.0 and 2.0 1.0 when the endpoint was the surviving fraction. These results confirmed that BPA was the most effective compound for the application of BNCT to the treatment of colon carcinoma. DISCUSSION We studied the induction of micronuclei in binucleated cells (CBMN) in a cell line of colon carcinoma. Micronuclei induction is an indicator of chromosomal damage caused directly by radiation or by DNA damage misrepair. In our studies the response to neutrons plus BOPP was similar to the response to treatment with the neutron beam alone for measured biologic effects, DNA damage, and surviving fraction. This finding can be explained if boron cell localization is taken into account. The porphyrin is more concentrated outside the cells, and the fraction that enters the cell is localized in the mitochondrial and lysosomal fractions (5). Monte Carlo simulations showed that for cells 10 mm in diameter when boron is localized outside the cell membrane the physical dose delivered to the nuclei is only 10% of that obtained when boron is distributed uniformly in the entire cell (16). Considering this information, the boron kerma calculated using the medium concentration is likely to be an overestimation of the absorbed boron dose to the cell nuclei, explaining the seemingly low effectiveness of BOPP. The CBE factors obtained therefore underestimate the actual boron RBEs (8). However, CBE factors remain the only meaningful option for calculating biologically weighted doses, because they intrinsically contain the effect of the microscopic nonuniformity. In this sense, they are different from
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Fig. 2. Distribution of the number of micronuclei (MN) per ARO81-1 binucleated cell in the different treatments. (A) Gamma radiation. (B) Thermal neutrons. (C) Neutrons + BOPP. (D) Neutrons + BPA. Average number of two independent experiments. BOPP = 2,4-bis-(a,b-dihydroxyethyl)-deutero-porphyrin; BPA = p-boronophenylalanine.
a pure RBE because they include the influence of cell morphology and boron average microdistribution on the actual dose delivered to cells.
Fig. 3. Survival of ARO81-1 cells under the different treatments. Data are fitted with a linear quadratic model. Each point is the average of six to eight wells SEM of two independent experiments. BOPP = 2,4-bis-(a,b-dihydroxyethyl)-deutero-porphyrin; BPA = p-boronophenylalanine.
In our studies we observed a small percentage of spontaneous DNA damage in control cells for the different treatments (no irradiation), which has also been reported in other tumor cell lines but not observed in normal cells (17). On the other hand, we can exclude that BPA and BOPP have genotoxic effects per se in the administered doses. These results agree with studies published elsewhere that have shown no cytotoxic or genotoxic effects for these boron compounds (18, 19). The dose–response curves obtained for the induction of micronuclei showed that both types of radiation (high and low LET) can be fitted with a sigmoid function. One explanation for this behavior may be that at low doses (approximately 2 Gy) the number of chromosome fragments can be low enough for them to have a high possibility of being expressed as a single MN each. At higher doses, the increased number of fragments can lead to two or more being incorporated into a single MN, thus explaining the leveling off in the yield of micronuclei. Furthermore, other authors attribute the depression in the induction of MN, as a function of dose, to the apoptotic process in an important number of cells (20). By contrast, our results show that the curves for low LET radiation (MN/BN; MNBN) can also be fitted with a linear function (R = 0.96) in agreement with other published results (21).
Biologic effectiveness factors of BNCT in colon carcinoma d M. A. DAGROSA et al.
Table 3. Experimental RBE and CBEs for colon carcinoma Parameter MN/BN % MN/BN Surviving fraction (MTT)
Endpoint level
Beam RBE
CBE BOPP
CBE BPA
0.8 1.2 43.4 52.0 65.6 75.8
2.6 0.7 4.2 1.1 2.6 0.7 4.4 1.1 2.4 0.6 2.3 0.6
4.6 1.1 8.0 2.2 2.9 1.1 5.1 1.9 2.0 1.0 1.9 1.0
12.2 2.3 19.6 3.7 12.3 2.3 17.8 3.6 3.5 1.2 3.7 1.3
Abbreviations: RBE = relative biologic effectiveness; CBE = compound biologic effectiveness; BOPP = 2,4-bis-(a,b-dihydroxyethyl)-deutero-porphyrin IX; BPA = p-boronophenylalanine; MN = micronuclei; BN = binucleated; MTT = 3-(4,5-dimetiltiazol-2il)-2,5-difeniltetrazolium bromide assay. Experimental RBE and CBE for colon carcinoma were calculated. In vitro cells were subject to four treatments. Two endpoints were measured: micronuclei induction and surviving fraction.
The reliability of the MNBN assay is controversial (22). It is known that reproductive cell death does not occur only before cell division, leading to overestimating the amount of cells that reach mitotic death because of the treatment. Also in some conditions (mostly for high doses of radiation), it can be difficult to distinguish between cells with only a nucleus from binucleated cells that have lost a nucleus after rupture caused by the manipulation of the samples, leading then to an underestimation of the actual number of binucleated cells. However, in our studies a good correlation between the induction of MN and the surviving fraction was found (R = 0.95, result not shown). As mentioned before, the calculated relative biologic effectiveness factors of the neutron beam (RBE) and of the boron compound (CBE) are required for estimating the biologically weighted dose delivered in BNCT, in our case for experimental colon carcinoma models. Each factor multiplies each of the components of the absorbed doses, thus allowing dose values to be expressed as biologically weighted dose. As it is known, these correction factors vary according to tissue composition and its specific radiosensitivity. In addition, RBE factors vary depending on the gamma and neutron spectra as a function of depth in tissue and dose levels,
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thus being different for each BNCT facility (23). Despite all the variables considered, the values obtained in this work are in the same range of those obtained by other authors in other tumor tissues and reactors (24, 25). Therefore, it is not surprising that RBE values for micronuclei induction are much higher than for cell inactivation when the values found elsewhere are taken into account, considering, though, that the cell line analyzed in this work is of tumoral origin and that RBE values depend strongly on the cell type. However, investigating the dose dependence of RBE rather than effect probability has major advantages, inasmuch as it is believed that features such as lesion formation, repair kinetics, and the assay chosen are factors that cancel out when taking the ratio of doses that produce the same level of effect (26). In fact, this work shows that the RBE hierarchy expected between chromosome aberration formation and cell killing is preserved. Special consideration must be taken in terms of transferring RBE values obtained from in vitro experiments to the in vivo situation, although this issue is of general concern in BNCT. Although tumor cells lack the typical high-level organization of normal tissues and so their response to radiation can be associated more easily with a parallel-organized tissue, which permits a more direct association with cell culture experiments, the main factor that obscures the direct transfer is the actual boron uptake in tumor cells. Several barriers must be crossed by a compound from the bloodstream until it reaches the target cell population (27). Colon carcinoma is the third most frequent malignant neoplasm in Western countries. After a complete resection, 5-year overall survival varies according to the stage at diagnosis (28). The development of new therapies is necessary. BNCT was applied for the treatment of liver metastases of colon carcinoma in a clinical setting at Pavia (6). Other groups started biodistribution studies of BPA and BSH in human patients with liver metastases of the same carcinoma (29, 30). The values obtained in these studies, to our knowledge, would be the first ones published for a model of experimental colon carcinoma in BNCT. It is also worth mentioning that these factors are the first experimental and biologic values obtained in the RA-3 facility.
REFERENCES 1. Coderre JA, Morris GM. The radiation biology of boron neutron capture therapy. Radiat Res 1999;1:1–18. 2. Busse PM, Harling OK, Palmer MR, et al. A critical examination of the results from the Harvard-MIT NCT program phase I clinical trial of neutron capture therapy for intracranial disease. J Neurooncol 2003;62:111–121. 3. Hill J, Kahl SB, Kaye A, et al. Selective tumor uptake of a boronated porphyrin in an animal model of cerebral glioma. Proc Natl Acad Sci U S A 1992;89:1785–1789. 4. Dagrosa MA, Viaggi ME, Jime´nez Rebagliati R, et al. Biodistribution of boron compounds in an animal model of undifferentiated thyroid cancer for boron neutron capture therapy. Mol Pharm 2005;2:151–156. 5. Pinelli T, Zonta A, Altieri S, et al. Taormina: From the first idea to the application to the human liver. In: Sauerwein W, Moss R,
6.
7. 8. 9.
Wittig A, editors. Research and development in neutron capture therapy. Bologna, Italy: Monduzzi Editore; 2002. p. 1065– 1072. Zonta A, Pinelli T, Prati U, et al. Extra-corporeal liver BNCT for the treatment of diffuse metastases: What was learned and what is still to be learned. Appl Radiat Isot 2009;67:S67– S75. Coderre J, Turcotte J, Riley K, et al. Boron neutron capture therapy: Cellular targeting of high linear energy transfer radiation. Technol Cancer Res Treat 2003;2:355. Santa Cruz GA, Zamenhof RG. The microdosimetry of the 10B reaction in boron neutron capture therapy: A new generalized theory. Radiat Res 2004;162:702–710. Fox J, Prise K. DNA lesions: Linear energy transfer and radiosensitive mutants. BJR Suppl 1992;24:48–52.
268
I. J. Radiation Oncology d Biology d Physics
10. Revell S. Radiation induced chromosome damage in man. In: Ishihara T, Sasaki M, editors. Relationship between chromosome damage and cell death. New York: Liss; 1983. p. 215–233. 11. Schweppe RE, Klopper JP, Korch C, et al. Deoxyribonucleic and profiling analysis of 40 human thyroid cancer cell lines reveals cross-contamination resulting in cell line redundancy and misidentification. J Clin Endocrinol Metab 2008;93:4331– 4341. 12. Kahl SB, Koo MS. Synthesis and properties of tetrakiscarboranecarboxylate esters of 2, 4-bis (-dihydroxyethyl) deuteroporphyrin IX. In: Allen BJ, Moore DE, Harrington BV, editors. Progress in neutron capture therapy for cancer. New York: Plenum Press; 1992. p. 223–226. 13. Dagrosa MA, Viaggi ME, Kreimann EL, et al. Selective uptake of p-borophenylalanine by undifferentiated thyroid carcinoma for boron neutron capture therapy. Thyroid 2002;12:7–12. 14. Miller M, Quintana J, Ojeda J, et al. New irradiation facility for biomedical applications at the RA-3 reactor thermal column. Appl Radiat Isotopes 2009;67:226–229. 15. Caria H, Chaveca T, Laires A, et al. Genotoxicity of quercentin in the micronucleus assay in mouse bone marrow erythrocytes, human lymphocytes, V79 cell line and identification of kinetochore-containing (CREST staining) micronuclei in human lymphocytes. Mutat Res 1995;343:85–94. 16. Gabel D, Foster S, Fairchild RG. The Monte Carlo simulation of the biological effect of the 10B (n,a) 7Li reaction in cells and tissues and its implication for boron neutron capture therapy. Radiat Res 1987;111:14–25. 17. Champion A, Hanson J, Court J, et al. The micronucleus assay: An evaluation of its use in determining radiosensitivity in vitro. Mutagenesis 1995;10:203–208. 18. Oliveira N, Castro M, Rodriguez A, et al. Evaluation of the genotoxic effect of the boron neutron capture reaction in human melanoma cells using the cytokinesis block micronucleus assay. Mutagenesis 2001;16:369–375. 19. Kinashi Y, Masunaga S, Ono K. Mutagenic effect of borocaptate sodium and boronophenylalanine in neutron cap-
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20. 21. 22. 23.
24.
25. 26. 27. 28. 29.
30.
ture therapy. Int J Radiat Oncol Biol Phys 2002;51:562– 567. Belyakov O, Prise K, Trott K, et al. Delayed lethality, apoptosis and micronucleus formation in human fibroblasts irradiated with X-rays or alpha-particles. Int J Radiat Biol 1999;75:985–993. Bedford J, Goodhead D. Breakage of human interphase chromosomes by alpha particles and X rays. Int J Radiat Biol 1989;55:211–216. Savage JRK. Micronuclei: Pitfalls and problems. Atlas Genet Cytogenet Oncol Haematol July 2000. Available at: http:// AtlasGenetics Oncology.org/Deep/MicronucleiID20016.html Gueulette J, Octave-Prignot M, De Costera B, et al. Intestinal crypt regeneration in mice: A biological system for quality assurance in non-conventional radiation therapy. Radiother Oncol 2004;73:148–154. Coderre J, Makar M, Micca P, et al. Derivations of relative biological effectiveness for the high-LET radiations produced during boron neutron capture irradiation of the 9L rat gliosarcoma in vitro and. in vivo. Int J Radiat Oncol Biol Phys 1993; 27:1121–1129. Suzuki M, Masunaga S, Kinashi Y, et al. The effects of boron neutron capture therapy on liver tumors and normal hepatocytes in mice. Jpn J Cancer Res 2000;91:1058–1064. Rossi HH, Zaider M. Microdosimetry and its applications. Berlin, Heidelberg, New York: Springer; 1996. p. 230. Jain R. Interstitial transport in tumors: Barriers and strategies for improvement. AACR 96th Annual Meeting 2005;108–113. Gravalos C, Garcia-Escobar I, Garcia-Alfonso P, et al. Adjuvant chemotherapy for stages II, III and IV of colon cancer. Clin Transl Oncol 2009;11:526–533. Wittig A, Malago M, Collete L, et al. Uptake of two 10B compounds in liver metastases of colorectal adenocarcinoma for extracorporeal irradiation with boron neutron capture therapy (EORTC Trial 11001). Int J Cancer 2008;122:1164–1171. Cardoso J, Nievas S, Pereira D, et al. Boron biodistribution study in colorectal liver metastases patients in Argentina. Appl Radiat Isot 2009;67:576–579.