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In vivo effect of chlorophyllin on γ-ray-induced sister chromatid

exchange in murine bone marrow cells

Article  in  Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis · April 1994

DOI: 10.1016/0165-1218(94)90085-X · Source: PubMed

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Mutation Research, 320 (1994) 329-334 329
© 1994 Elsevier Science B.V. All rights reserved 0165-1218/94/$07.00

MUTGEN 1981

In vivo effect of chlorophyllin on y-ray-induced sister chromatid exchange

in murine bone marrow cells
P. M o r a l e s - R a m l r e z * and M.C. Garcla-Rodrlguez
Instituto Nacional de Investigaciones Nucleares, Sierra Mojada 447, Lomas Barrilaco, CP 11010, Mexico, D.F., Mexico
(Received 26 March 1993)
(Revision received3 September 1993)
(Accepted 9 September 1993)

Keywords: Chlorophyllinradioprotection; Gamma-rays; Sister-chromatid exchange; Mouse bone marrow

Summary

The aim of the present work is to determine the radioprotective capacity of chlorophyllin, by
measuring the reduction of y-ray-induced sister-chromatid exchange (SCE) in murine bone marrow cells
in vivo. The results obtained in two separate experiments, using 10, 50 and 100/zg of chlorophyllin per g
of body weight (bw), indicate that chlorophyllin per se did not have any effect on the SCE frequency and
that the dose of 1 0 0 / x g / g bw protects 100% against the induction of SCE by 1.0 Gy of y-rays; 5 0 / z g / g
bw protects less than 50% and 1 0 / z g / g bw affords no protection.

As a consequence of the demonstration of the Renner, 1990; Elias et al., 1990; R6scheisen et
mutagenic and carcinogenic effect of radiation al., 1991). In a previous study, evidence was ob-
and chemical agents, the possibility of obtaining tained indicating that the antimutagenic capacity
substances capable of reducing these effects has of some vegetable extracts was proportional to
been explored. First, evidence of substances that the chlorophyll content (Lai et al., 1980). Both
protect cells against the effect of radiation expo- chlorophyll and its more soluble sodium and cop-
sure was demonstrated; such chemicals are more per salt, chlorophyllin, demonstrated a clear an-
effective when they act as radical scavengers timutagenic capacity in the Ames test (Ong et al.,
(Garriot and Crowe, 1983; Murray et al., 1988a,b, 1986; Negishi et al., 1989) as well as antitrans-
1989, 1990; Miyazaki et al., 1990). forming activity in vitro (Wu et al., 1990).
As a result of widespread interest in chemical Recently, evidence has shown that chloro-
mutagenesis, agents with antimutagenic activities phyllin also has radioprotective capacity in the
were sought especially in natural products, in- S M A R T system in Drosophila melanogaster
cluding some dietary sources (Hayatsu et al., 1988; (Zimmering et al., 1990).
Sister-chromatid exchange (SCE), first re-
ported by Taylor in 1958, has been amply studied
* Cooresponding author. Fax 521-37-98. since then, especially after the development of

SSDI 0165-1218(93)E0112-2
330

techniques based on bromodeoxyuridine (BrdU) Animals

incorporation into DNA (Latt, 1973; Perry and Two- to three-month-old B A L B / c male mice
Wolff, 1974). The latter, in turn, allowed a more weighing approximately 30 g were used in the
accurate analysis of this phenomenon. experiments. Animals were kept in plastic cages
SCE is regarded as a good index of D N A with sawdust bedding, under controlled tempera-
damage (Latt et al., 1981). Although ionizing ture and dark-light conditions, and fed Purine
radiation was not initially considered efficient at chow and water ad libitum.
inducing SCE (Perry and Evans, 1975; Littlefield
et al., 1979), evidence has been obtained that Administration of chemicals
ionizing radiation is capable of inducing SCE BrdU (Sigma) previously adsorbed to activated
both in vitro (Abramovsky et al., 1978; Chao and charcoal was administered intraperitoneally in an
Rosenstein, 1984) and in vivo (Morales-Ramlrez aqueous suspension (Morales-Ramirez, 1980).
et al., 1983), and either in cells with their D N A The dose was 1.5 m g / g bw of BrdU.
unsubstituted (Renault et al., 1982; Chao and Chlorophyllin (Sigma) was administered in
Rosenstein, 1984; Morales-Ramirez et al., 1984a) aqueous solution intraperitoneally, at doses of
or unifilarly BrdU-substituted (Perry and Evans, 1 0 - 1 0 0 / x g / g bw 4.5 h before BrdU treatment.
1975; Morales-Ramirez et al., 1983). Moreover, it
has been shown that in murine bone marrow cells Irradiation
in vivo, the SCE increase induced by y-rays per- For the y-ray treatment, animals were exposed
sists 96 h after exposure (Morales-Ramirez et al., to a 6°Co source (Vick Rad) with a dose of 1.0 Gy
1984). Recent evidence has indicated that all and dose rate of 2.6 G y / m i n . To confirm the
gamma-ray-induced lesions related to SCE for- exposure dose in all irradiated animals, capsules
mation are persistent and capable of eliciting with four TLD-200 dosimeters (Harshaw) were
SCE at the same locus in successive cell divisions placed in the leg near each femur.
(Morales-Ramlrez et al., 1988).
The aim of the present work is to determine Bone marrow cell metaphases
the radioprotective capacity of chlorophyllin, by Colchicine was administered intraperitoneally
measuring the reduction of y-ray-induced sister in aqueous solution (7.0 /xg/g bw) 22 h after
chromatid exchange in murine bone marrow cells BrdU treatment. Animals were killed by cervical
in vivo. This same system was previously used to dislocation 2 h later and the marrow ceils were
prove the radioprotective capacity of cysteamine obtained by injecting a buffered saline solution at
(Mendiola and Morales, 1989). one end of the previously dissected femurs.
The cell suspension was centrifuged at 100 × g
Materials and methods for 10 min. The pellet was resuspended with 0.3
ml of buffered saline solution and treated with
Protocol 5.0 ml of 0.075 M KCI at 37°C for 15 min. Then
To determine the radioprotective capacity of the cell suspension was centrifuged 10 min and
chlorophyllin, four groups of at least four mice the pellet resuspended in methanol-acetic acid
each were formed. The control group was treated 3:1 for 15 rain; this process was repeated three
with 1.5 m g / g bw of BrdU (Morales, 1980) at the times. Finally, the fixed cell suspension was
beginning of the experiment, and with colchicine dropped on clean and chilled slides; when neces-
7 / x g / g bw 22 h later. In addition to these treat- sary the slides were dried under the flame.
ments, the other groups were also exposed to The differential staining of sister chromatids
either chlorophyllin 4.5 h before BrdU adminis- was achieved by a modification (Goto et al., 1975)
tration (chlorophyllin group), or to 1.0 Gy of of the fluorescence plus Giemsa method (Perry
y-rays 30 min before BrdU (irradiated group) or and Wolf, 1974). Briefly, the slides were treated
to both at the times indicated (chlorophyllin- with a 10 -4 M aqueous solution of the fluo-
irradiated group). Each experiment was repeated rochrome Hoechst 33258 for 10 min in the dark,
once. washed with water and exposed to a black light
 331

lamp at a distance of 1.0 cm for 1 h. Then they 100 -4D

C
were treated with a saline solution in citrate U
M
buffer at 60°C for 15 min, washed with warm U
L
80

water and finally stained with 5% Giemsa in A

T
phosphate buffer for 30 min. v
I 60
E

Scoring F
R 40
E
The SCE scoring was carried out in 30 well Q
U
differentially stained metaphases per mouse. The E 20
N
mitotic index was determined by scoring the num- C
Y O~
ber of metaphases per 2000 cells. The average
1 2 3 4 5 6 7 8 9 10
generation time was measured using the method SCE / CELL
of Ivett and Tice (1982), by quantifying the num- Fig. 1. Cumulative frequencies of cells (%) with respect to the
ber of cells in first, second or third division per SCE number in murine bone marrow cells treated with noth-
100 metaphases. ing ( + ), 100 tzg/g bw chlorophyllin (*), irradiated 1.0 Gy ( [] )
The statistical evaluation was done with Dun- or 100/xg/g bw chlorophyllin and 1.0 Gy of ~,-rays(x).
nett's test for several groups and different sample
sizes (Cheung and Holland, 1992). SCE per cell; this increase is reproducible and
significant ( a = 0.01). With respect to the effect
Results
of chlorophyllin on the irradiated animals, the
Table 1 shows the pooled results obtained in data indicate that the dose of 100 / z g / g bw
two separate experiments, using 10, 50 and 100 protects 100% against the induction of SCE by
/ x g / g bw of chlorophyllin. No significant differ- 1.0 Gy of y-rays; 50 / x g / g bw protects less than
ences between the data of these experiments 50% and 1 0 / z g / g of bw affords no protection.
were observed. Chlorophyllin per se did not have Fig. 1 shows the effect of chlorophyllin on SCE
any effect on the SCE frequency. The exposure to induction by y-rays in the cell populations. This
1.0 Gy of y-rays (1.07 + 0.16 Gy according to effect was attained by analyzing the SCE frequen-
T L D ) induced an increase of approximately one cies in the cell population resulting from pooling
all the cells analyzed in each group of mice; then
TABLE 1 the cumulative frequency in percentage versus
E F F E C T OF C H L O R O P H Y L L I N D O S E ON SCE INDUC-
the number of SCE per cell was plotted. This
T I O N BY y-RAYS IN M O U S E BONE M A R R O W CELLS figure shows that y-radiation causes an increase
IN VIVO in cells with higher frequencies of SCE with re-
spect to the control cells. Thus, in the control
Group ChlorophyllinRadiation SCE/cell n population only 50% of the cells have more than
(/zg/g) (Gy) (x_+SD) three SCEs; in the irradiated ones, these ceils
Control 0 0 3.4+0.18 15 represent 70% of the total population. Chloro-
Chlorophyllin 100 0 3.5 + 0.23 8 phyllin per se did not cause any change. How-
50 0 3.6_+0.20 8 ever, the chlorophyllin pre-treatment prevented
10 0 3.6_+0.26 8 the effect caused by the radiation exposure.
Radiation 0 1 4.5_+0.12 * 14 The results obtained with respect to average
generation time ( A G T ) for the same treatments
Chlor-Rad 100 1 3.5_+0.27 ** 8
50 1 4.0_+0.37 8 are shown in Table 2. These results represent the
10 1 4.5 _+0.24 * 7 pooled data from two separate experiments which
were not significantly different. These data indi-
n, number of mice.
cate that chlorophyllin reduces A G T , even in the
* Significant results vs. control group, Dunnett's test, p <
0.01. chlorophyllin-irradiated group. Because of this, it
** Significant results irradiated vs. chlorophyllin-irradiated was not possible to measure the effect of chloro-
groups, Dunnett's test, p < 0.01. phyllin on radiation-induced cell cycle delay.
332

TABLE 2 To explore the possibility that a lower dose of

EFFECT OF C H L O R O P H Y L L I N AND y-RAYS ON THE chlorophyllin prevents the reduction on MI
CELL A V E R A G E G E N E R A T I O N TIME IN M U R I N E caused by radiation, experiments were made us-
BONE M A R R O W IN VIVO ing 1 and 5 /zg/g of bw doses, in animals that
were not exposed to BrdU. The results indicate
Group Chlorophyllin Radiation AGT(h) n
(/xg/g) (Gy) (x_+ SD)
that there was no protective effect (data not
shown).
Control 0 0 12.1 + 0.16 15

Chlorophyllin 100 0 10.2_+0.84 * 8 Discussion

50 0 9.7_+0.60 * 8
10 0 10.3_+0.22 * 8
The data obtained in the present study con-
Irradiated 0 1 12.6_+0.33 14 firm the radioprotective capacity of chlorophyllin,
Chlor-Irrad 100 1 10.8_+0.31 * 8 previously observed in the SMART system in
50 1 10.9_+0.75 * 8 Drosophila (Zimmering et al., 1990). The dual
10 1 10.5+0.60 * 7 capacity of chlorophyllin as an antimutagen (Lai
n, number of mice.
et al., 1980; Ong et al., 1986; Kimm et al., 1982;
* Significant results, Dunnett's test, p < 0.01. Terwel and Van der Hoeven, 1985) and as a
radioprotectant (Zimmering et al., 1990) is inter-
esting because it is a biological molecule and
As shown in Table 3, with regard to the radio- seems to be non-toxic.
protective effect of chlorophyllin measured by the Ong et al. (1986) indicated that the antioxida-
mitotic index (MI), no significant effect of chloro- tive and ion scavenger properties of chlorophyllin
phyllin per se on MI was observed. However, it could determine its antimutagenic capacity by
was noted that chlorophyllin protects the cells reacting with either the active groups or the radi-
against the 25% reduction in MI caused by radia- cals of the agents. In the case of its radioprotec-
tion exposure. Complete radioprotection was ob- rive capacity, it is probable that this is due to the
served with the lowest dose ( 1 0 / x g / g bw). These ion scavenger capacity.
results represent the pooled data from two sepa- Besides suggesting the radioprotective capacity
rate experiments which were not significantly dif- of chlorophyllin, the results obtained in this study
ferent. show that this substance can act in vivo in mam-
mals. The fact that a protective capacity has also
TABLE 3 been observed in murine spermatogonia in vivo
E F F E C T OF C H L O R O P H Y L L I N ON MITOTIC I N D E X (manuscript in preparation) indicates that after
R E D U C T I O N CAUSED BY y-RAYS IN M U R I N E BONE administration chlorophyllin is widely distributed
M A R R O W CELLS IN VIVO in the organism and actively incorporated by the
cells.
Group Chlorophyllin Radiation MI a n
The low toxicity of chlorophyllin, its appar-
(/zg/g) (Gy) (x± SD)
ently general distribution when administered to
Control 0 0 38.9 + 4.5 15
the organism, and its high efficiency as a radio-
Chlorophyllin 100 0 40.3_+3.5 8 protectant, open the possibility of its use in hu-
50 0 40.1_+3.5 8 man beings, in circumstances in which radiation
10 0 39.8_+2.9 8
exposure is expected, e.g., radioactive decontami-
Irradiated 0 1 29.3_+3.8 * 14 nation activities.
Chlor-lrrad 100 1 40.1 _+1.9 8
50 1 41.1_+3.7 8 Acknowledgements
10 1 44.3 _+3.5 7
We wish to thank Jorge Mercader M., Angel
a Mitotic index, number of metaphases per 1000 cells.
n, number of mice.
Reyes P., Perfecto Aguilar, V., Felipe Beltrfin B.
* Significant results, Dunnett's test, p < 0.01. and Enrique Fernfindez V. for their excellent
 333

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Miyazaki, T., Y. Hayakawa, K. Suzuki, M. Suzuki and M.
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