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Sistema de Información Científica
Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
Rev. Int. Contam. Ambient. 8 (2). 73-76, 1992
GENOTOXIC EFFECTS OF OVATIFOLIN ACETATE, A SESQUITERPENE
LACTONE ISOLATED FROM
Podanthus ouatifolius
Lag.
(Compositae)
Mario ALARCON, Giselind WEIGERT, Soledad DUK and María de los Angeles GARCIA
Department of Molecular Biology. Faculty of Biological Sciences and Natural Resources. P.O. Box 2407. Ap. 10. Concepción,
Chile, Universidad de Concepción
(Recibido enero 1992, aceptado octubre 1992)
Keywords: antineoplastic effects, ovatifolin, sesquiterpene lactone, genotoxicity, micronucleus test
ABSTRACT
Ovatifolin acetate (OA), a sesquiterpenic lactone has been isolated from
Podanthus ova-
tifolius
Lag. Compositae. When tested in standard tests in KB cells of human epidermoid
nasopharingeal carcinoma, it showed antineoplastic activity. Since these drugs are well
known to induce genetic damage the genotoxic activity of OA was evaluated using the
micronucleus test in mice with male Balb/c mice. OA showed a cytotoxic effect by modifying
cell proliferation kinetics of mouse bone marrow erythrocytes and also increased micronu-
cleated cells.
RESUMEN
Se ha aislado acetato de ovatifolina (AO), una lactona sesquitergnica de
Podanthus ova-
tifolius
Lag. Compositae, planta chilena; ella muestra propiedades antineoplásicas en la
prueba estándar de células KB, epidermoides de carcinoma nasofaríngeo humano. Siendo
los antineoplásicos inductores potenciales de un amplio daño genético, se valora la acción
genotóxica del acetato de ovatifolina mediante la prueba de micronúcleos, en médula ósea
de ratones machos
Mus musculus,
Balb/c. A0 se muestra como un poderoso agente citotó-
xico capaz de alterar la cinetica de proliferación o maduración de los eritrocitos en médula
ósea de ratón, que así mismo produce un incremento de celulas micronucleadas.
INTRODUCTION
More and more drugs for cancer therapy have been
developed during the last years, but an xdesirable
genotoxic effect on normal cells is however frequently
present (Bhakuni
et al.
1976, Jenssen and Ramel 1980,
Mac Gregor
et al.
1987, Cea
et al.
1990). For this reason
chemotherapeutic treatment would only be recommen-
ded when the risk-benefit evaluation makes it appro-
priate.
Genotoxic agents are capable of causing a variety of
cell nuclear changes, such as micronuclei in mouse
bone-marrow polychromatic ery throcytes (PCE) (Wild
1978, Hart
et al.
1983, Hayashei
et al.
1984, Alarcón
et al.
1986, Mirkova 1987). They are generally accepted
to reflect clastogenicity.
OA, a sesquiterpene lactone isolated from natural ex-
tracts of
Podanthus ovatifolius,
exhibits antineoplastic
properties as tested in standard KB human epidermoid
carcinoma of nasopharynx and PS (P 338 lyinphocytic
leukemia) assay procedures.
A program for screening of antitumoral effects of
chemicals isolated from chilean plants, has been going
on for the last decades (Gnecco
et al.
1973, Bhakuni
et al.
1976, Cea
et al.
1990).
This agent (OA) and others were tested in our labora-
tory for their genotoxic activity (Alarcón
et al.
.
1986,
Cea
et al.
1990). Based on this study it is necessary to
make a clinical determination to see whether this com-
pound is convenient as an antitumoral drug.
M. Alarcón
et
al.
MATERIALS AND METHODS
OA was supplied by the Phytochemistry Lab. Univer-
sity of Concepción. The cytotoxicity test for
KB
cells
and
in
viuo PS test were carried out at the National
Cancer Institute (NCI), USA.
Two month old male Balb/c mice (Molecular Bio-
logv Department Biotherium, University of Concep-
ción) weighing ca. 20 g were intraperitoneally injected
with a single OA dose (volume 0.2 ml) diluted in
dioxan-distilled water (1
:
10).
Four doses were selected (1.25,2.5,5.0 and 10.0 mg/kg)
on the basis of the data with
KB
cells LD 50 (2.50 mg/l
of TC medium).'Doxorubicine (Adriamycine, Farmi-
talia) at 10.00 mg/kg was used as positive control and a
mixture of dioxane distilled water as negative control.
Dioxan-water was selected as a solvent, since the OA is
insoluble in water and other solvents tested. The dilu-
tion was 1:10 because in this way dioxan was less toxic
and it was the lowest dilution which disolved the hig-
hest dose of OA.
Four mice per dose were sacrified 30 h after injec-
tion and femurs were removed. Femurs were prepared
for the bone-marrow micronucleus test as proposed by
Schmid (1975) but modified according to Oliver and
Goldstein (1978) and Das and Kar (1980). Slides were
stained with May-Grünwald and Giemsa solutions ac-
cording to the schedule outlined by Cole et al. (1979)
which maximized the differenciation between polych-
romatic (PCE) and normochromatic (NCE) erythrocy-
tes. An average of 5000-9000 PCE and NCE per animal
per dose were scored from coded slides and micronu-
cleated polichromatic (MPCE) and normochromatic
(MNCE) erythrocytes were recorded.
The Mann-Whitney U test was employed for statis-
tical analysis.
TABLE 1. AVERAGE VALUES AND STANDARD DEVIATIONS OF QUANTITATIVE
ANALYSIS OF BONE-MARROW ERYTHROCYTES FROM MICE TREATED
WITH OVATIFOLIN ACETATE
DOSES
mg/Kg
MPCE/1000 PCE
MNCE/I000 NCE
PCE/NCE
DOXO
27.57f 4.33
5.78f 1.45
0.58f 0.06
10.00
D-W
1:lO
;
PCE
=
polychromatic erythrocytes
NCE
=
normochromatic erythrocytes
MPCE
=
micronucleated polychromatic erythrocytes
MNCE
=
miaonucleated normochromatic erythrocytes
DOXO
=
doxorubicine
D-W
=
dioxan-water
OA =ovatifolin acetate
GENOTOXICITY OF OVATIFOLIN ACETATE
TABLE
11.
MANN-WHITNEY U TEST RESULTS OF BONE-MARROW ERYTHROCYTES
FROM MICE TREATED WITH OVATIFOLIN ACETATE
~~
MPCE/IOOOPCE
MNCE/IOOONCE
PCE/NCE
D-W
U=O
U=O
U=O
1:lO
p=0.014
p=0.014
p=0.014
A
A
>
U=O
U=l
U=O
U=5
U=6
U=O
1.25
p=0.014 p=0.029
p=0.014 p=0.243
p=0.343 p=O.OI 4
A
>
A
A
u=o
u=o
u=o
u=o
u=o
U=O
u=3
u=o
u=o
2.50
p=0.014 p=0.014 p=0.014
p=0.014 p=0.014 p=0.014
p=O. 100 p=0.014 p=0.014
A
>
A
>
>
>
A
A
U=O
U=8
U=O
U=O
U=O
U=7
U=O
U=O
LJ=O
U=O
U=O
U=3
5.00
p=0.014 p=0.557 p=0.014 p=0.014
p=0.014 p=0.443 p=0.014 p=0.014
p=0.014 p=0.014 p=0.014 p=O.100
A
>
A
A
A
>
A
A
A
U=O
U=O
U=O
U=O
U=O
U=l
U=3
U=6
U=O
U=5
U=3
U=O
U=O
U=8
U=l
10.00
p=0.014 p=0.014 p=0.014 p=0.014 p=0.014 p=0.029 p4.100 p=0.343 p=0.014 p=0.213 p=O.100 p=0.014 p=0.014 p=0.557 p=0.029
A
A>A
A
A
>
A
A
>
DOSIS
DOXO D-W
1.25
2.50
5.00
DOXO D-W
1.25
2.50
5.00
DOXO D-W
1.25
2.50
5.00
10.00
1
:
10;
10.00
1:lO;
10.00
1
:
10;
The
>
indicaies ihat the effect o1 the lefi-column dose is greater ihan [he correspondeni effect o1 botiom-coluinn dose
The A indicaies ihat ihe effeci of botto,m-column dose is greaier ihan [he correspondeni effect of lefi-columri dose
MPCE
=
micronucleated polychromaiic eryihrocyies
MNCE
=
micronucleated normochromatic eryihrocytes
PCE
=
polichromatic eryihrocyies
NCE
=
normochromatic eryihrocytes
DOXO
=
doxorubicine
D-W
=
dioxan-water
RESULTS AND DISCUSSION
The data of the mouse bone-marrow study are presen-
ted in Table
1.
Table
11
shows the "U" and asocciated
p values as compared one to one with the effects of
different doses by the Mann-Whitney "U" test.
OA significantly increases the MPCE incidence at
dosages of 1.25 and 2.50 mg/kg, but at 5.00 and 10.00
mg/kg the incidences o£ MPCE decreased significati-
vely below the frequency induced by dioxan-water mix-
ture (negative control). On the other hand, the MNCE
frequency increased significantly at 2.50 mg/kg and
then decreases at higher OA doses.
The PCE/NCE ratio decreased at 2.50 mg/kg and
reached the lowest values at 5.00 mg/kg coincidently
with the MPCE and MNCE frequencies peaks.
The negative control showed unexpectedly high MPCE
frequency. Since negative controls are usually water or
saline solutions, we suspect that our results are due
to dioxan itself.
The micronuclei observed were both round and oval-
shaped with sizes ranging between 1/5
-
1/8 of the
cell diameter, which according to Heddle and Carrano
(1977) and Yamamoto and Kikushi (1980) confirms the
clastogenic origin o£ the MN. On the other hand,
the bone-marrow data show a remarkable depression
and it is evident that it became flooded with peripheral
blood at al1 OA doses used.
The induction of micronucleated cells provides a
sensitive measure of chromosome breakage although
not al1 types of chromosome aberrations become mi-
cronuclei (Hayashei et
al.
1984). Short term in
vivo
assays such as the micronucleus test do reflect the com-
plex pharmacokinetics involved in the uptake, meta-
bolism and distribution of genotoxic agents (Jenssen
and Ramel 1980). Nevertheless, a closer approach to
evaluate the genetic-risk of the organism would be
to analyze not only the micronuclei formation in bone
marrow but also other nuclear damage in the tissue of
actual interest, because the tissue-response to clastoge-
nic injury could be tissue-specific (Goldberg
et
al.
1983,
Proudlock and Allen 1986).
The
in
vitro
LD,, of OA for KB cell LD,, is 2.50
mg/l TC medium (National Cancer Institute USA).
Considering this as a reference, at an empirically equi-
valent dose of 2.50 mg/kg OA induced the formation
M. Alarcón
et al.
of typical micronuclei. At 1.25 and 2.50 mg/kg OA
shows a high clastogenic activity in mouse bone-marrow
cells. At higher dosages this effect disappears due to
the increased cell lethality observed by both the his-
tological analysis and an anatomical inspection of the
bone-marrow. The PCE/NCE ratio decreased below
0.5
in mice treated with 2.5-10.0 mg/kg
OA
indicating im-
paired proerythroblast-polychromatic erythrocyte fluxe
or polychromatic-normochromatic erythrocyte tran-
sition.
These results showed that OA is a cytotoxic agent with
ability to modify the proliferation of bone-marrow cells
and is a clastogenic agent capable of inducing micro-
nuclei in mouse bone-marrow polychromatic ery thro-
cytes.
ACKNOWLEDGEMENTS
Supported by Dirección de Investigación, Universi-
dad de Concepción, Proyecto 20.3 1.23. OA wcre kindly
supplied by Dr. Mario Silva, Phytochemistry Labora-
tory, Universidad de Concepción.
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