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Received 22 August 2001; received in revised form 14 November 2001; accepted 14 November 2001
Abstract
The genotoxic effects of malathion was evaluated using chromosome aberration, sister chromatid exchange (SCE) and
sperm abnormality assays in mice. All the three acute doses (2.5, 5 and 10 mg/kg) of malathion tested in the present study,
induced significant dose-dependent increase in the frequency of chromosome aberrations and sperm abnormalities, but did not
affect the total sperm count. The highest acute dose induced a >12-fold increase in the frequency of chromosome aberrations,
two-fold increase in the frequency of SCEs and four-fold increase in the frequency of sperms with abnormal head morphology
following intraperitoneal (i.p.) exposure. Further, a significant increase in the frequency of SCEs was observed, but the increase
was not dose-dependent. At higher doses, malathion induced a moderate delay in cell cycle as evident from the increase in
average generation time (AGT). The present findings suggest that technical grade malathion is a potent genotoxic agent and
may be regarded as a potential germ cell mutagen also. © 2002 Elsevier Science B.V. All rights reserved.
Keywords: Malathion; Organophosphorus insecticide; Chromosome aberration; Sister chromatid exchange; Sperm abnormality; Replication
index
1383-5718/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 1 3 8 3 - 5 7 1 8 ( 0 1 ) 0 0 3 4 1 - 2
224 S. Giri et al. / Mutation Research 514 (2002) 223–231
other insecticides are reported to exhibit higher levels a closely inbreed colony. The animals were housed
of chromosome aberrations and SCEs [12]. Malathion in polypropylene cages (4–5 animals per cage with
has been reported to produce chromosome aberrations sawdust as the bedding material) at 25 ± 5 ◦ C tem-
in plants also [13]. However, negative results in assays perature on a 12 h light/dark cycle. The animals were
for dominant lethals in mice [14]; chromosome loss supplied with dry food pellets commercially available
and non-disjunction in male germ cells [15], somatic (Hindustan Liver Ltd., New Delhi), and water ad libi-
mutations in white–zeste eye spot test [16], and wing tum. Healthy animals of both sexes, in the age group
spot test [17] in Drosophila melanogaster have also of 10–12 weeks were used for the experiments.
been reported.
Most of the studies on the mutagenic effects of 2.3. Dose
malathion are supported from in vitro studies [3,18–
21]. However, in vivo test is especially relevant The highest sub-lethal acute dose (10 mg/kg) used
for genotoxicity, because it allows consideration of in the present study was standardized by trial. The
factors of in vivo metabolism, pharmacokinetics, animals tolerated the highest dose with minimal toxic
and DNA-repair process and is also useful in further symptoms. The toxic symptoms were mostly neuro-
investigation of mutagenic effects detected by an in logical. However, the animals recovered within 1 h of
vitro genotoxicity test [22]. In the present study, the the treatment. MMC (2 mg/kg) was used as a positive
genotoxic potential of malathion, as revealed by bone control, and the untreated control mice were given
marrow chromosome aberration, SCEs and sperm equal volumes of normal saline only.
shape abnormality assays in mice is reported.
2.4. Chromosome aberration assay
Table 1
Frequency of chromosome aberrations in the bone marrow cells of mice induced by malathiona,b
Dose (mg/kg) Route Fixation Total Chromatid Isochromatid Exchanges SCU Total % Aberration
time (h) cells/n aberrationsc (mean ± S.D.)
Gaps Breaks Gaps Breaks
aberrations were classified as indicated in Table 1. Fifty well spread second division metaphases iden-
Gaps have not been considered for statistical analysis tifiable by their uniform differential staining pattern
due to their controversial genetic significance [23]. containing 40 chromosomes were analyzed for SCE
per animal. Every switch of staining between the
2.5. Sister chromatid exchange (SCE) assay sister chromatids was scored as one SCE.
In addition to SCEs, cells were analyzed for the
SCE assay was done as described by Goto et al. [24]. relative frequency of first division metaphases (M1,
Ten minute before the various treatments, the mice identifiable by uniform staining of both the sister chro-
weighing between 20 and 22 g were sub-cutaneously matids), second division metaphases (M2, identifiable
implanted with bromodeoxy uridine (BrdU) tablets by differential staining of the sister chromatids), and
(50 mg) under light ether anesthesia. Before sacrifice, third and subsequent division metaphases (M3, iden-
the animals were treated with colchicine (4 mg/kg, tifiable by non-uniform pattern of staining). These
i.p.) for one and half hours. The animals were sac- information were used to evaluate cell proliferation
rificed by cervical dislocation after 24 h of the treat- kinetics as described by Tice and Hollaender [25].
ment; both the femora were dissected out and cleaned Replication index (RI) or proliferation index is the
of any adhering muscles. Metaphase spreads were average number of replications completed by meta-
prepared as described above and staining was done as phase cells and is calculated as follows:
described by Goto et al. [24]. In brief, the slides were
treated for 10 min with Hoechst 33258 (50 g/ml) in 1 × (% first division metaphases) + 2
dark at room temperature. The slides were then rinsed ×(% second division metaphases) + 3
in distilled water, mounted in 2× SSC (NaCl—sodium ×(% subsequent division metaphases)
citrate, pH = 6.8) and kept under direct sun light in RI =
100
moist condition for 30–40 min depending upon the
intensity of sun light. Then the slides were rinsed and The average generation time (AGT) was determined
stained in 3% buffered Giemsa (pH = 7.0) for 6 min. as the ratio of BrdU duration (h) and RI.
226 S. Giri et al. / Mutation Research 514 (2002) 223–231
2.6. Sperm abnormality assay frequencies of gaps were observed for all the doses
(2.5, 5 and 10 mg/kg) tested. A tentative assessment
Five equal sub-divisions of each dose were succes- of the distribution of breaks and gaps revealed that
sively injected (i.p.) at 24 h intervals to healthy and the distal regions of the long chromosomes were more
sexually matured male mice. The controls were given vulnerable to malathion.
an equal volume of normal saline. Animals treated Malathion induced a significantly higher frequency
with MMC (2 mg/kg) served as positive controls. of aberrations (P < 0.001) for all the three doses
The animals were sacrificed by cervical dislocation tested at both the time points (24 and 48 h) as com-
35 days after the first injection. Both the cauda epi- pared to the control (Table 1). The dose response
didymis were dissected out and placed in 1 ml of PBS curve at 24 h of i.p. treatment (Fig. 2A) shows a
(pH = 7.2). The sperms were released by mechanical linear increase in the frequency of aberrations with
disruption and washing of the epididymis. The suspen- increasing dose (r = 0.9734, P < 0.05). Although
sion was sieved through two layers of musolin cloth to the frequency of aberrations in the p.o. route was
remove the tissue debris. A drop of the suspension was
taken on a clean slide and a smear was made, air-dried
and fixed in absolute methanol for 10 min. The slides
were stained in the following day in 0.1% eosin Y
for 8–10 min. One thousand sperms per animal were
scored and the abnormalities were categorized as close
as to those described by Wyrobek and Bruce [26].
Sperm count was done as described by Rai and
Vijayalaxmi [27]. In brief, an aliquot (0.05 ml) from
the sperm suspension (1 ml) was diluted (1:40) with
PBS. A sample was taken in the Neubauer chamber
and the total sperm count in eight squires of 1 mm2
each was determined. The cell number was multiplied
by 5 × 104 and the sperm number was expressed as
cells/epididymis.
3. Results
Table 2
Effect of malathion on cell cycle kinetics and the frequency of SCEs in the bone marrow cells of micea
Dose TM M1 M2 M3 % M1 % M1 RI AGT AGT Total SCE/M SCE/M
(mg/kg) (mean ± S.D.) (mean ± S.D.) cells (mean ± S.D.)
Control 181 18 154 9 9.94 10.46 ± 2.18 1.950 12.30 12.36 ± 0.12 50 2.80 3.06 ± 0.26
156 14 135 7 8.97 1.955 12.27 50 3.08
185 24 152 9 12.97 1.918 12.51 50 3.32
MMC (2.0) 240 91 146 3 37.91 42.09 ± 4.04 1.633 14.69 14.99 ± 0.28 50 9.16 9.30 ± 0.13
187 86 095 6 45.98 1.572 15.26 50 9.34
210 89 117 4 42.38 1.595 15.04 50 9.42
2.5 157 14 138 5 08.91 11.26 ± 2.63 1.942 12.35 12.59 ± 0.28 50 5.94 5.97 ± 0.21∗∗∗
195 21 169 5 10.76 1.917 12.51 50 5.78
163 26 134 3 14.11 1.858 12.91 50 6.20
5 166 24 139 3 14.45 15.41 ± 3.28 1.873 12.81 12.84 ± 0.20∗ 50 6.24 6.06 ± 0.17∗∗∗
173 22 147 4 12.71 1.895 12.66 50 6.04
152 29 119 4 19.07 1.835 13.07 50 5.90
10 185 35 142 3 18.91 1.772 13.07 50 6.24
170 29 138 3 17.05 1.847 13.54 50 5.90
205 39 162 4 19.02 18.32 ± 1.10∗∗ 1.829 12.99 13.21 ± 0.28∗∗ 50 6.36 6.16 ± 0.23∗∗∗
a
Control: only normal saline was given. TM: total metaphases; AGT: average generation time; M: metaphase; RI: replication index;
SCE: sister chromatid exchange. Statistical analysis: Student’s t-test (n = 3).
∗ Significantly different from control (P < 0.05).
∗∗ Significantly different from control (P < 0.01).
∗∗∗ Significantly different from control (P < 0.001).
relatively lower than the i.p. route, but these were (Table 2). However, no significant dose response could
not significant (P > 0.05). Similarly, except for the be found for SCEs in the present study. The AGT was
highest dose (10 mg/kg, P < 0.01), no significant significantly increased following 5 mg/kg (P < 0.05)
difference could be found in the time response study and 10 mg/kg (P < 0.01) of malathion exposure. The
(Fig. 2B). Further, the sub-acute (2 mg/kg for 5 days) frequency distribution of SCEs did not show any large
treatment produced lower effects (P < 0.01) than the variation as most of the cells showed SCEs in the range
corresponding acute dose (10 mg/kg) (Table 1). of 2–8 SCEs per cell (data not shown).
All the three acute doses (2.5, 5 and 10 mg/kg) of Following 2.5, 5 and 10 mg/kg of malathion treat-
malathion, induced significantly higher (P < 0.001) ment, significant increases (P < 0.02, 0.001 and
frequency of SCEs as compared to the control value 0.001, respectively) in the frequency of sperm head
Table 3
Incidence of sperm shape abnormality induced by malathion and total sperm count in micea,b
Dose (mg/kg) No. of sperms No. of % Abnormal sperms Sperm count/epididymis
studies per animal abnormal sperms (mean ± S.D.) (mean ± S.D.) ×106
Control 3000/3 53 1.76 ± 0.35 6.42 ± 0.18
MMC (2.0) 3000/3 314 10.46 ± 0.65 5.16 ± 0.23
2.5 3000/3 89 2.96 ± 0.41∗ ac 6.44 ± 0.25
5 3000/3 158 5.20 ± 0.40∗∗ ab 6.21 ± 0.24
10 3000/3 227 7.56 ± 0.35∗∗ bc 6.22 ± 0.15
a Control: only normal saline was given. Statistical analysis: Student’s t-test.
b Values having similar letters are significantly different from each other (P < 0.02 (a, b); P < 0.001 (c)).
∗ Significantly different from the control (P < 0.05).
∗∗ Significantly different from the control (P < 0.01).
228 S. Giri et al. / Mutation Research 514 (2002) 223–231
abnormalities were noted as compared to the un- lite(s) only. Therefore, the possibility of a cumulative
treated control (Table 3). The amorphous type of head effect even for a shorter duration may not be ruled out.
morphology was more prevalent than other types. SCEs arise from reciprocal exchanges of DNA at
However, no significant difference could be observed apparently identical loci of the sister chromatids of
in the total number of sperms per epididymis as a duplicated chromosome in response to a damaged
observed in the sperm count assay (Table 3). DNA template [25,29]. It is suggested that there are at
least two forms of SCE induction. One is by damaging
DNA [30] and other is by inhibiting DNA synthesis
4. Discussion [31]. In eukaryotic cells, the frequency of SCEs is
reported to be increased following exposure to geno-
Malathion is a widely used pesticide with high toxic agents that induce DNA damage capable of
potential for human exposure. The genotoxic data interfering with DNA replication, but not strand
have been somewhat inconclusive with regard to breakage only [29]. The significant increase (P <
malathion exposure. It is found to be non-genotoxic in 0.001) in SCEs following malathion exposure in the
a variety of test systems [14–17]. Positive results have present study further supports the genotoxic poten-
also been reported by many authors [3,9–12]. The tial of malathion. Malathion induces SCEs in other
results of the present study indicate that malathion is test systems also [9]. Several studies have reported
genotoxic in mice. that malathion is a very potent cell cycle inhibitor
Malathion induced a variety of chromosomal [10–12,32]. In the present study, malathion induced a
aberrations in the bone marrow cells (Table 1). The small but significant delay in cell cycle in the 5 mg/kg
dose-dependent increase in the frequency of chro- (P < 0.05) and 10 mg/kg (P < 0.01) treated group,
mosome aberrations as observed presently (Fig. 2A), but not in the 2.5 mg/kg treated group. Further, we
indicate that the chemical might be available in greater failed to find any dose-dependent increase in the fre-
concentrations to the target cells at higher doses. The quency of SCEs. Our findings are in agreement with
significantly higher frequency of aberrations observed earlier studies reporting lack of correlation between
at 48 h of the treatment could be due to residual ef- the frequency of SCEs and dose [18,33]. We have ob-
fect of the chemical or its toxic metabolite(s). Similar served a clear dose-dependent increase in chromosome
effects have been observed for carbofuran also [28]. aberrations in the present study (Fig. 2A) in contrast
In addition, for 2.5 and 5 mg/kg treated groups, no to those reported earlier [11,34,35]. Therefore, the
significant difference in the frequency of chromo- cell cycle delay may only be partially responsible for
some aberrations at 24 and 48 h of the treatment was the lack of dose response observed here for SCEs, but
found (Fig. 2B). This indicates that not only the par- other factors may also be involved. The significance of
ent compound, but also its secondary metabolite(s) this finding is difficult to explain at present. However,
may induce mutagenic effects. Malaxon, the oxida- it appears that malathion may induce chromosome
tion product of malathion is reported to be mutagenic aberrations and SCEs through different mechanisms.
in human peripheral blood lymphocytes in vitro [21]. These findings merit further investigations.
However, the significant difference (P < 0.01) in Sperm abnormality assay is a sensitive and reliable
the time response study in the groups treated with endpoint and is widely used to identify germ cell
10 mg/kg could be due to death and removal of highly mutagens [25–27,36]. Induction of abnormal sperms
damaged cells from the population. is presumed to be a result of naturally occurring errors
In the sub-acute (2 mg/kg for 5 days) treatment in the differentiation process, or the consequence of an
group, significantly lower frequency of chromosome abnormal chromosome complement [37]. The charac-
aberrations was observed as compared to the equiv- teristics controlling sperm head shape are carried on
alent acute dose (10 mg/kg) (Table 1). However, the the autosomes and sperm abnormality test identifies
higher frequency of chromosome aberrations in this those agents, which cause small alterations in testic-
group than the acute dose of 2.5 (P < 0.01) and ular DNA [38,39]. The significant increase in sperm
5 mg/kg (P < 0.01) cannot be explained in the light head abnormality (Table 3) induced by malathion in-
of residual effect and/or secondary genotoxic metabo- dicates its probable potential as a germ cell mutagen.
S. Giri et al. / Mutation Research 514 (2002) 223–231 229
It has been reported that exposure to pesticides results and may be regarded as a potential germ cell mutagen.
in a decrease in sperm count [40,41]. However, in the Therefore, application of malathion that exposes large
present study, malathion in the dose ranges tested did populations should be restricted.
not show any change in sperm count as compared to
the control.
The exact mechanism of the genotoxic effects of Acknowledgements
malathion is not known. Organophosphorus insecti-
cides containing the P=S bond (called “thion”) are We are thankful to the Head of the Department of
converted to P=O (called “oxon”) by a system of Life Science, Assam University, Silchar, for providing
enzymes called microsomal mixed-function oxidases laboratory facilities.
(MFO) in which the enzyme cytochrome P-450 play
a major role [42]. The oxons are highly toxic com-
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