Mutagenesis vol.16 no.5 pp.369–375, 2001
Evaluation of the genotoxic effects of the boron neutron capture
reaction in human melanoma cells using the cytokinesis block
micronucleus assay
N.G.Oliveira1,2, M.Castro2,3, A.S.Rodrigues1,4,
I.C.Gonçalves5, R.Cassapo1, A.P.Fernandes5,
T.Chaveca1,2, J.M.Toscano-Rico3 and J.Rueff1,6
1Department
of Genetics, Faculty of Medical Sciences, New University of
Lisbon, R. da Junqueira 96, P 1349-008 Lisbon, 2Faculty of Pharmacy,
University of Lisbon, Lisbon, 3CFEC, Faculty of Medicine, University of
Lisbon, Lisbon, 4University Lusófona, Lisbon, and 5Nuclear and
Technological Institute, Portuguese Research Reactor, Sacavém, Lisbon,
Portugal
The present work reports on the genotoxicity of the boron
neutron capture (BNC) reaction in human metastatic
melanoma cells (A2058) assessed by the cytokinesis block
micronucleus assay (CBMN) using p-borono-L-phenylalanine (BPA) as the boron delivery agent. Different concentrations of BPA (0.48, 1.2 and 2.4 mM) and different
fluences of thermal neutrons were studied. Substantial
genotoxic potential of α and lithium particles generated
inside or near the malignant cell by the BNC reaction was
observed in a dose–response manner as measured by the
frequency of micronucleated binucleated melanoma cells
and by the number of micronuclei (MN) per binucleated
cell. The distribution of the number of MN per micronucleated binucleated cell was also studied. The BNC reaction
clearly modifies this distribution, increasing the frequency
of micronucleated cells with 2 and, especially, ≥3 MN and
conversely decreasing the frequency of micronucleated cells
with 1 MN. A decrease in cell proliferation was also
observed which correlated with MN formation. A discrete
genotoxic and anti-proliferative contribution from both
thermal neutron irradiation and BPA was observed and
should be considered secondary. Additionally, V79 Chinese
hamster cells (chromosomal aberrations assay) and human
lymphocytes (CBMN assay) incubated with different concentrations of BPA alone did not show any evidence of
genotoxicity. The presented results reinforce the usefulness
of the CBMN assay as an alternative method for assessment
of the deleterious effects induced by high LET radiation
produced by the BNC reaction in human melanoma cells.
Introduction
The capture of thermal neutrons (nth, low energy, average
value 0.025 eV) by the minor stable isotope of boron (10B)
releases α and lithium particles. This reaction is called the
boron neutron capture (BNC) reaction [10B(n,α)7Li: 10B ⫹
nth→[11B]→4He (α) ⫹ 7Li ⫹ 2.3 MeV]. The propagation of
α and 7Li particles in biological tissues is characterized by a
short range (~9 and 5 µm, respectively) and a high linear
energy transfer (LET) with a remarkable destructive power
(reviewed in Hawthorne, 1993; Coderre and Morris, 1999).
This reaction has thus been primarily proposed for the treatment
of some types of malignant melanoma and glioma in a
6To
targeted binary radiation therapy called boron neutron capture
therapy (BNCT).
The present work reports on the genotoxicity induced by
the BNC reaction in human metastatic melanoma cells (A2058)
assessed by the cytokinesis block micronucleus assay (CBMN
assay) using p-borono-L-phenylalanine (BPA) (Figure 1) as
the boron delivery agent. Different concentrations of BPA as
well as different periods of irradiation with thermal neutrons
were studied.
The CBMN assay is a standard method for assessing DNA
damage and has been reported as a useful biomarker for
evaluation of the genotoxic effects of ionizing radiation (Brooks
et al., 1990; Fenech et al., 1990; Ono et al., 1996; Shibamoto
et al., 1998; Streffer et al., 1998; Gil et al., 2000a). Formation
of micronuclei (MN) is also known to be associated with
mitosis-linked cell death (reviewed in Cohen-Jonathan et al.,
1999).
The CBMN assay has been studied in different cell types
(Fenech, 2000), either normal or tumoral, including melanoma
cells (Champion et al., 1995; Courdi et al., 1995; Widel et al.,
1997; Poma et al., 1999). Fenech (2000) has extensively
reviewed the inherent advantages of the CBMN assay and a
broad range of different applications.
The possible intrinsic genotoxic and anti-proliferative contributions of the two components of the BNC reaction, the
thermal neutrons and the boron delivery agent BPA, were also
studied. Neutron capture reactions involving other nuclides,
such as hydrogen [1H(n,γ)2H] and nitrogen [14N(n,p)14C], as
well as proton recoils from the fast neutrons [1H(n,n’)1H), are
present in the final reaction (Gupta et al., 1994) and can
be assessed by the thermal neutron irradiation of non-BPA
incubated cells. The same is valid for the background of γ-rays
from the reactor, which can be physically monitored. This low
LET radiation increases the genotoxicity of the BNC final
reaction, as it is a function of irradiation time, and its
contribution can also be evaluated by comparison with genotoxicity curves of 60Co γ-rays.
BPA is an adequate 10B delivery agent for melanoma BNCT
(Coderre et al., 1987, 1991; Mishima et al., 1989a,b; Matalka
et al., 1993; Mishima, 1997) and is selectively accumulated
in melanoma cells since there is an elevated amino acid
transport rate at the cell membrane (Coderre and Morris,
1999). Moreover, BPA is currently being tested in various
clinical trials not only for malignant melanoma but also for
other types of cancer, namely glioblastoma multiforme. BPA,
as previously shown through in vitro and in vivo experiments
(Coderre et al., 1992; Matalka et al., 1994), is also selectively
accumulated in other non-melanoma tumoral cells presently
having a key role in BNCT. Concerning this point it seems
relevant to evaluate the genotoxic effects of BPA not only
in A2058 human melanoma cells (CBMN assay) and its
contribution to the total effect of the BNC reaction in these
cells, but also in non-tumoral cells, namely V79 Chinese
whom correspondence should be addressed. Fax: ⫹351 21 3622018; Email: [email protected]
© UK Environmental Mutagen Society/Oxford University Press 2001
369
N.G.Oliveira et al.
for 10 min. For each experimental point 1000 binucleated (BN) A2058 cells
with well-preserved cytoplasm were scored. MN were identified according to
the criteria of Caria et al. (1995) using a 1250⫻ magnification on a
light microscope. Two indices were evaluated, number of micronuclei per
binucleated cell (MN/BN), which represents the average number of MN
per BN cell, and frequency of micronucleated binucleated melanoma cells
(%MNBN), which represents the fraction of cytokinesis blocked (BN) cells
with MN, regardless of the number of MN per BN cell.
The decrease in cell proliferation for the experiments described above was
assessed using the frequency of BN cells (%BN). For this index 1000 human
melanoma cells with well-preserved cytoplasm were analyzed according to
number of nuclei using a 500⫻ magnification.
Fig. 1. Chemical structure of the boron delivery agent BPA.
hamster cells (chromosomal aberrations assay, CA assay) as
well as in human lymphocytes (CBMN assay).
Materials and methods
Chemicals and culture medium
Fetal calf serum, RPMI medium, Ham’s F-10 medium, cytochalasin B (Cyt-B),
L-glutamine, penicillin and streptomycin were purchased from Sigma (St Louis,
MO). 4-Borono-L-phenylalanine 10B enriched (⬎99%) was obtained from
KatChem (Prague, Czech Republic). Acetic acid, methanol and Giemsa dye
were obtained from Merck (Darmstadt, Germany). Phytohaemagglutinin (PHA;
HA 15) was purchased from Murex (Dartford, UK) and reconstituted in 5 ml
of sterile water. Heparin was obtained from Braun (Melsungen, Germany).
Colchicine was purchased from Fluka (Buchs, Switzerland) and trypsin from
Difco Laboratories (Detroit, MI).
Melanoma cell culture and BPA incubation
Human melanoma A2058 cells (~2.0⫻105) were cultured in RPMI medium
supplemented with 10% fetal calf serum, penicillin (100 IU/ml) and streptomycin (100 µg/ml) and incubated at 37°C under an atmosphere containing 5%
CO2. This cell line was previously established from a brain metastasis of a
43-year-old man (Todaro et al., 1980) and was kindly provided by Dr O.Csuka
(Budapest).
For the BPA-treated cells medium supplemented with a BPA stock solution
was prepared with a final BPA concentration of 2.4 mM (500 µg/ml, 24.0
p.p.m. 10B). Cells were seeded in 25 cm2 tissue culture flasks (Greiner,
Frickenhausen, Germany) and incubated either with 5 ml of BPA medium
(0.48, 1.2 and 2.4 mM) or with BPA-free culture medium. The cells were
grown as monolayers for 48 h and then irradiated with thermal neutrons.
Thermal neutron irradiation protocol
Irradiation of human melanoma cells took place at the vertical access of the
thermal column of the Portuguese Research Reactor (RPI). Characterization
of the radiation field and reduction of the background γ-radiation from the
reactor were essential tasks for these radiobiological experiments and have
been described elsewhere (Gonçalves et al., 1999). The neutron spectrum is
basically thermal, with a low epithermal component and reduced γ-ray
background. The thermal neutron flux (φth), epithermal neutron flux (φepi) and
γ-ray dose rate in air (Dγ, air) are of the order of 5.7⫻107 nth/cm2/s1, 2⫻104
nepi/cm2/s1 and 0.2–0.3 Gy/h, respectively.
Three different periods of irradiation were studied, 30, 60 and 120 min,
corresponding to average fluences (⫾ SD) of 1.1 ⫾ 0.06, 2.2 ⫾ 0.01 and 4.3
⫾ 0.30⫻1011 nth/cm2 as measured by individual dosimetry using gold foil
activation. Two independent experiments were performed. Controls included
cells irradiated with thermal neutrons without BPA incubation (neutron control
cells), cells incubated with BPA without thermal neutron irradiation (BPA
control cells) and cells unirradiated and without BPA incubation (background
control cells). BPA control cells were used to assess the genotoxic potential
of BPA as compared with background control cells using a two-tailed Student’s
t-test for statistical analysis.
CBMN assay in A2058 human melanoma cells
For the CBMN assay 6 h after the irradiation with thermal neutrons the cell
culture medium was removed, the cells washed and placed in BPA-free culture
medium. Cyt-B was added at a final concentration of 6 µg/ml (Van Hummelen
and Kirsh-Volders, 1990) and the cells were grown for a further 22 h for
recovery of binucleated A2058 cells. The cells were then harvested by
trypsinization, rinsed and submitted to mild hypotonic treatment as described
elsewhere (Van Hummelen and Kirsh-Volders, 1990; Gil et al., 2000a,b). The
centrifuged cells were placed on dry slides and smears were made. After air
drying the slides were fixed with cold methanol (30 min). One day later the
slides were stained with Giemsa (4% v/v in 0.01 M phosphate buffer, pH 6.8)
370
Evaluation of BPA genotoxicity in human lymphocytes: CBMN assay
Aliquots of 500 µl of whole blood from four healthy donors were cultured in
4.5 ml of Ham’s F-10 medium supplemented with 24% fetal calf serum,
penicillin (100 IU/ml), streptomycin (100 µg/ml), 1% L-glutamine and 1%
heparin (50 IU/ml). Lymphocytes were stimulated using 25 µl of PHA and
incubated at 37°C. At 24 h culture the cells were exposed to different doses
of BPA (0.12, 0.24, 0.36 and 0.48 mM) for 3 h. In these experiments BPA
was not previously dissolved in the culture medium, as for the A2058 and
V79 Chinese hamster cell experiments. Instead, BPA was prepared as a stock
solution of 48.1 mM dissolved in 0.2 M HCl. Background control cells were
incubated with the maximum quantity of 0.2 M HCl used for the 0.48 mM
BPA concentration (this volume did not exceed 1%). After BPA incubation
the cells were centrifuged and placed in fresh culture medium. At 44 h culture
Cyt-B was added (6 µg/ml). At 72 h culture cells were harvested by
centrifugation, treated twice with 5 ml of a mixture of RPMI 1640:deionized
water 4:1 (pH 7.2) supplemented with 2% fetal calf serum and submitted to
a mild hypotonic treatment as described above. Preparation of slides and
scoring of MN was also performed as described for melanoma cells in 1000
BN lymphocytes and %MNBN, which represents the fraction of cytokinesis
blocked (BN) cells with MN was assessed. In order to evaluate cell proliferation, 1000 human lymphocytes cells were analyzed using a 500⫻ magnification.
Statistical analysis for evaluation of the genotoxic potential of BPA was
carried out using a two-tailed Student’s t-test.
Evaluation of BPA genotoxicity in V79 Chinese hamster cells: CA assay
Wild-type V79 Chinese hamster (MZ) cells were kindly provided by Prof.
H.R.Glatt (Mainz and Postdam). These cells were cultured in the same culture
medium used for human melanoma cells and incubated at 37°C under an
atmosphere containing 5% CO2. A BPA stock supplemented culture medium
was prepared with a final BPA concentration of 2.4 mM (500 µg/ml, 24.0
p.p.m. 10B).
V79 cells (~1.0⫻105) were seeded in 25 cm2 tissue culture flasks (Greiner)
and incubated with either 5 ml of BPA medium (0.48, 1.2 and 2.4 mM) or
BPA-free culture medium. The cells were grown as monolayers in this medium
for 64 h. The medium was then removed and colchicine added in BPA-free
culture medium at a final concentration of 0.6 µg/ml. Cells were grown for a
further 2.5 h and then harvested by trypsinization. After a 3 min hypotonic
treatment with 75 mM KCl at 37°C the cells were fixed with methanol/acetic
acid (3:1) and slides prepared and stained with Giemsa (4% v/v in 0.01 M
phosphate buffer, pH 6.8) for 10 min. Six independent experiments were
carried out for each BPA concentration as well as for untreated cultures and
100 metaphases were observed using a 1250⫻ magnification and a light
microscope. Scoring of the different types of aberrations followed the criteria
described by Rueff et al. (1993). Mitotic index (MI) was estimated by scoring
1000 V79 cells using a 500⫻ magnification under a light microscope.
Statistical analysis for evaluation of the genotoxic potential of BPA was
carried out using a two-tailed Student’s t-test.
Results
Figure 2 shows the genotoxic effects of the BNC reaction in
human melanoma cells preincubated for 48 h with three
different concentrations of BPA (0.48, 1.2 and 2.4 mM) and
irradiated with thermal neutrons for three periods of time, 30,
60 and 120 min, corresponding to average fluences of 1.1, 2.2
and 4.3⫻1011 nth/cm2. In this figure two important indices
concerning the CBMN assay are represented, MN/BN (average
⫾ SD) (Figure 2A) and %MNBN (average ⫾ SD) (Figure 2B).
The human melanoma cell line used (A2058) has a high
background frequency of MN compared with normal cells
(⬎10-fold), namely human lymphocytes (Gil et al., 2000b).
This inherent characteristic, which reflects a high level of
Genotoxicity of the boron neutron capture reaction
Fig. 3. Distribution of the number of micronuclei per micronucleated
binucleated human melanoma cell incubated with BPA (2.4 mM) and
irradiated with thermal neutrons (average values of two independent
experiments).
Fig. 2. Induction of micronuclei in human melanoma cells incubated with
BPA (0.48, 1.2 and 2.4 mM) and irradiated with thermal neutrons. (A) MN/
BN. (B) %MNBN. Results are expressed as means ⫾ SD from two
independent experiments.
spontaneous DNA damage, has been reported for other human
melanoma cell lines (Champion et al., 1995; Poma et al., 1999).
Regarding both indices, MN/BN and %MNBN, a dose–
response relationship was observed for both BPA concentration
and thermal neutron fluence. Some differences may be
observed, namely in the shapes presented by the curves. The
MN/BN curves present a linear dependence on fluence whereas
the %MNBN curves suggest some kind of saturation for the
higher fluence studied, especially for the two highest BPA
concentrations.
The number of MN per micronucleated BN cell is also an
important indicator of the degree of lesion induced by the
BNC reaction. In Figure 3 the relative frequency of each type
of micronucleated cell classified by number of MN (1, 2, 3,
4, ⬎4) is represented for the three fluences studied as well
as for micronucleated BPA control cells (2.4 mM). These
micronucleated cells have essentially 1 or 2 MN per BN cell
(~80 and 15%, respectively), a pattern that is also present in
the human melanoma control cells (~84 and ~11%). However,
thermal neutron irradiation of BPA-incubated cells distinctly
modified the number of MN per micronucleated cell. For the
higher thermal neutron irradiation period the frequency of
micronucleated cells with 1 MN decreased to values of ~45%,
whereas the frequency of micronucleated cells with 2 and 3
MN increased (~25 and 15%, respectively). A clear dosedependent relationship was found for the frequency of micronucleated cells with 3 MN, which seems to be a good indicator
of the extent of DNA damage. In addition, heavily damaged
cells containing ⬎3 MN were observed, especially at the
higher irradiation level (~15%).
A dose-dependent decrease in %BN was observed for human
Fig. 4. Decrease in the frequency (%) of binucleated human melanoma cells
incubated with BPA (0.48, 1.2 and 2.4 mM) and irradiated with thermal
neutrons. Results are expressed as means ⫾ SD from two independent
experiments.
melanoma cells incubated with the two highest concentrations
of BPA (1.2 and 2.4 mM) and irradiated with thermal neutrons
(Figure 4). Regarding each thermal neutron fluence studied, a
dose-dependent decrease in %BN was also observed with
increasing BPA concentrations. For the higher fluence studied
and for the higher BPA concentration this proliferation index
decreased to ~50% of control cells. In addition, a very good
correlation (r ⫽ –0.98) was found comparing the decrease in
%BN with the increase in %MNBN for irradiated melanoma
cells incubated with 2.4 mM BPA.
Thermal neutron irradiation in the absence of BPA incubation
resulted in a slight increase in both MN/BN and %MNBN
(Figure 2A and B). For the highest fluence studied these
indices presented approximately double the values (2.1-fold)
compared with background melanoma cell controls. The number of MN per micronucleated BN cell shows a pattern similar
to background cells and BPA control cells. The micronucleated
cells usually have 1 or 2 MN and the fluence of thermal
neutrons did not significantly affect this index. Thermal neutron
irradiation itself reduced %BN by ~10% but not in a dosedependent manner (Figure 4).
Concerning the genotoxic and anti-proliferative effects of
BPA in human melanoma cells, BPA incubation of nonirradiated cells (BPA control cells) mildly increased both
371
N.G.Oliveira et al.
excluding gaps (%CAEG) were observed in V79 Chinese
hamster cells for the same concentrations of BPA (0.48, 1.2
and 2.4 mM) as used in melanoma cells and with a long
incubation period of ⬎60 h (Figure 5B). The average %CAEG
presented by negative control cultures (1.5 ⫾ 0.5%) was within
the normal values presented by this cell line in previous
experiments (Oliveira et al., 1997; Alves et al., 2000). BPA
incubation increased this background by only 0.8% at the
maximum level (0.48 mM BPA; NS). No evidence of any
anti-proliferative effects due to BPA incubation was observed.
In addition, human lymphocytes incubated with BPA (0.12–
0.48 mM) for a 3 h period (G1 phase) did not show evidence
of genotoxicity using the CBMN assay, as shown in Figure
5C. The %MNBN values presented by negative control cultures
(0.58 ⫾ 0.29%) were within the normal range found in healthy
donors (Caria et al., 1995; Gil et al., 2000b; Oliveira et al.,
2000). No evidence of any anti-proliferative effects due to
BPA incubation was observed.
Discussion
Fig. 5. Genotoxic and anti-proliferative effects of BPA without thermal
neutron irradiation in different cell types. (A) Human melanoma cells
(CBMN assay, six independent experiments). (B) V79 Chinese hamster cells
(CA assay, six independent experiments). (C) Human lymphocytes (CBMN
assay, four healthy donors). Results are means ⫾ SD. CAEG, chromosomal
aberrant cells excluding gaps; BN, binucleated cells, MNBN,
micronucleated binucleated cell; MI, mitotic index. *P ⬍ 0.05.
indices (MN/BN and %MNBN) and decreased %BN as compared with the background values. These results are presented
in detail in Figure 5A. For the highest BPA concentration
studied (2.4 mM) the MN/BN index was 0.126 ⫾ 0.031 for
BPA control cells, whereas 0.097 ⫾ 0.016 MN/BN was found
as the background value (NS, P ⫽ 0.08). %MNBN was 9.9
⫾ 1.8 for BPA control cells versus 7.6 ⫾ 1.3 for melanoma
background cells (P ⬍ 0.05). A value of P ⫽ 0.05 was found
for 0.48 mM BPA (%MNBN of 9.5 ⫾ 1.7).
V79 Chinese hamster cells (CA assay) and human lymphocytes (CBMN assay) incubated with different concentrations of
BPA alone showed no evidence of genotoxicity. No significant
increases in the frequencies of chromosomal aberrant cells
372
The promising usefulness of BNCT in the treatment of some
types of malignant melanoma (Mishima et al., 1989a,b; Busse
et al., 1997; Mishima, 1997) has prompted a multi-disciplinary
effort in order to improve its efficacy. The cytotoxic effects
of the BNC reaction have been extensively evaluated using
clonogenic assays (percent tumor cell survival) either using
in vitro or in vivo approaches (reviewed in Coderre and Morris,
1999). There are also some reports using the MN assay as the
end-point for study of this reaction. These reports have been
mainly performed using an experimental mouse squamous cell
carcinoma model (SCCVII cells) in C3H/He mice (Ono et al.,
1996; Masunaga et al., 1999). Additional data on the pattern
of genotoxicity induced by this high LET radiation towards a
highly invasive human melanoma cell line (A2058), using the
CBMN assay, is thus important to help in understanding the
mechanisms underlying cell death/damage.
The MN/BN index roughly represents the average number
of chromosomal lesions present in a BN human melanoma
cell and %MNBN represents the frequency of DNA damaged
cells regardless of the number of lesions per cell. Both indices
are a measure of the genotoxic burden caused by the BNC
reaction and are complementary. Although %MNBN increases
in a typical dose-dependent manner, the genotoxicity observed
in terms of MN/BN is higher. The different shapes of the two
curves can be explained by the increase in the number of
α-particles produced per cell, with no parallel increase in the
number of cells damaged. The increase in multi-micronucleated
cells, which enhances the MN/BN index, is also in agreement
with a similar increase in cell cycle arrest (as measured by the
BN index), especially for the two highest BPA concentrations tested.
Mechanistic knowledge on DNA and cell damage induced
by α-particles remains limited (National Research Council,
1999). Due to the high LET nature of this radiation, traversal
of cells by α-particles is considered to be lethal. The insult
from α-particles is concentrated in a relatively small densely
ionizing track that crosses the cell in ⬍10–12 s and deposits a
large localized energy (~10–50 cGy). In addition, individual
damage is mainly due to direct ionization more than to
hydroxyl radical reactions (National Research Council, 1999).
Clonogenic lethality for α-particles has been extensively
described and it has been well established that high LET
Genotoxicity of the boron neutron capture reaction
radiation produces more biological damage per unit absorbed
dose than sparsely ionizing radiation, such as X- and γ-rays
(National Research Council, 1999). Dose-dependent chromosomal damage has also been studied and associated with the
deleterious effects of α-radiation. In fact, α-particles induce
CAs (Edwards et al., 1980; Lloyd et al., 1988; Bauchinger
and Schmid, 1998; Pohl-Rülling et al., 2000), MN (Bilbao
et al., 1989; Brooks et al., 1990; Ono et al., 1996; Belyakov
et al., 1999) and sister chromatid exchanges (Aghamohammadi
et al., 1988; Nagasawa and Little, 1992; Lehnert and Goodwin,
1997). Recent in vitro studies with α-particle emitters have
shown that up to 80% of cells traversed by one α-particle
survive damage, yet sustain a considerable increase in mutation
frequency (Hei et al., 1997). Traversal of cells by up to four
α-particles further increases mutation frequency, while having
a moderate cytotoxic effect. Additionally, other studies have
shown that the biological effects of α-radiation are not limited
to cells actually traversed. Enhanced frequencies of mutagenic
effects have been observed in non-irradiated ‘bystander’ cells
(Nagasawa and Little, 1992; Zhou et al., 2000), including
chromosomal instability (Lorimore et al., 1998; Kadhim et al.,
1994), possibly through reactive oxygen species (Narayanan
et al., 1997; Lehnert and Goodwin, 1997; Wu et al., 1999).
Concerning this point, we have measured the concentration of
malondialdehyde in the supernatants of melanoma cell cultures
exposed to the BNC reaction and have found no difference
from unirradiated controls (data not shown).
Monte Carlo calculations for the BNC reaction suggested
that ~10% of the total dose is due to external 10B, 45% from
10B present in the cytoplasm and 45% from the nucleus
(reviewed in Fairchild et al., 1990). Accordingly, the results
presented here could also be the result of genotoxicity due to
α-particles generated in the cytoplasm or even in the extracellular compartment.
The BNC reaction has also proven to interfere with cell
cycle progression as a function of BPA concentration and
thermal neutron fluence. Delays in cell cycle progression, with
the purpose of providing time for DNA repair and also for
induction of transcription of genes that could enhance DNA
repair (reviewed in Elledge, 1996), have been unequivocally
related to checkpoint disturbances. Low LET ionizing radiation
has been shown to induce cell cycle arrest in G1 before DNA
replication, in S phase and also in G2 before mitosis. Inhibition
of cdk–cyclin complexes, which regulate both the G1/S and
G2/M checkpoints, mediate these arrests (reviewed in CohenJonathan et al., 1999). High LET radiation such as α-particles
induces more extensive delays in the S or G2 phases as
compared with low LET radiation, although Azzam et al.
(2000) have shown the existence of a p53-mediated G1 arrest
in human diploid fibroblasts exposed to α-particles.
Thermal neutron irradiation of non-BPA-incubated cells
slightly increased the yields of MN/BN and %MNBN as a
function of fluence. This effect, which approximately duplicates
the values of both indices for the fluence of 4.3⫻1011 nth/cm2,
is mainly due to the γ-ray background from the reactor and
other reactions following the interaction of thermal neutrons
with biological tissues. In a 2 h irradiation period, which
corresponds to the highest fluence studied, the cells were
exposed to ~0.4–0.6 Gy of γ-rays. The CBMN assay was
performed under the same experimental conditions as described
for the BNC reaction but with 60Co γ-rays (data not shown)
and the linear fitting parameters of the MN/BN and MNBN
(as decimal fraction) curves were 0.120 ⫾ 0.007 and 0.080 ⫾
0.005 per Gy, respectively (γ-ray dose rate 31.01 mGy/min,
dose range 0.25–3 Gy). The γ-ray background dose of 0.4–0.6
Gy thus results, by extrapolation, in values of 0.05–0.07 for
MN/BN and 3.2–5.0 for %MNBN.
The BPA contribution to the genotoxic burden of the BNC
reaction was also assessed and shown to be secondary, although
we cannot exclude that BPA alone is genotoxic to human
melanoma cells. Nevertheless, no dose–response dependence
for BPA genotoxicity nor a substantial increase in %MNBN
values was observed. The results obtained with V79 Chinese
hamster cells (CA) and with human lymphocytes (CBMN) do
not show any kind of genotoxicity and/or anti-proliferative
effects even at high doses (120 µM–2.4 mM). These two cell
types, with their inherent sensitivities and different genetic
end-points, were used in order to achieve a maximal likelihood
of properly ascertaining a possible genotoxic activity of BPA
alone. The results are in agreement with the idea that boron
compounds are not positive in the usual mutagenicity tests
performed (reviewed in Commission of the European Community, 1993; World Health Organization, 1998), although
only a few studies of this nature are available, mainly with
inorganic boron compounds.
The rationale for the use of BPA in BNCT has been the
assumption that this compound belongs to the class of melanin
precursors as it is an analog of phenylalanine. This argument
is still a matter of debate (Hawthorne, 1993) and other
mechanisms could be involved, namely chemical complex
formation of BPA with melanin monomers (Mishima, 1997).
In addition, active transport of this compound through the
melanoma cell membrane and that of other non-melanoma
tumors has been observed (reviewed in Coderre and Morris,
1999) and recent data on the underlying mechanisms of this
transport published (Wittig et al., 2000). These observations
suggest utilization of the compound in other metabolic processes in tumoral cells but the reason why non-melanoma
cells selectively accumulate BPA remains unknown (Wittig
et al., 2000).
The slight increase in %MNBN and decrease in %BN on
BPA treatment herein observed for human melanoma cells
could be partially explained by the chemical resemblance to
phenylalanine. In fact, the cytotoxicity and genotoxicity of
melanin synthesis precursors have long been known for melanoma cells (reviewed in Herlyn and Houghton, 1992) and some
reports have been published concerning this matter (Miranda
et al., 1997; Poma et al., 1999). However, other mechanisms
could also be involved, since in other non-melanoma tumors,
e.g. SCCVII cells from mice treated with BPA, the MN
frequencies were also slightly higher (~2-fold) than those
observed in the control group (Masunaga et al., 1999).
An overall analysis of the presented results reveals a
substantial genotoxic potential associated with the BNC reaction due to the generation of α and lithium particles inside or
near the malignant cell. Discrete genotoxic contributions from
both thermal neutron irradiation alone and the boron delivery
agent BPA alone were observed and should be considered
secondary.
Finally, it should be pointed out that the interest of this
reaction is far beyond its application in malignant melanoma
and glioma. Other tumors are significantly destroyed by this
reaction and the reaction could be used to treat rheumatoid
arthritis in so-called boron neutron capture synovectomy
(reviewed in Hawthorne, 1998). Moreover, one can look at
the BNC reaction as a useful tool in understanding the
373
N.G.Oliveira et al.
mechanisms of high LET α-particle radiation-induced DNA
damage in tumoral and normal cells.
Acknowledgements
We warmly thank our colleague Prof. A.Laires for his many helpful suggestions,
fruitful observations and pertinent criticisms. Our appreciation is extended to
Dr A.Ferro de Carvalho for the 60Co γ-irradiation as well as Dr A.Ramalho
and Eng. F.Cardeira from the RPI. We also gratefully acknowledge our
colleague O.Monteiro Gil for technical assistance.
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Received February 13, 2001; accepted April 6, 2001
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