# Clastogenic Effects of Glyphosate in Bone Marrow Cells of Swiss Albino Mice

Authors: Sahdeo Prasad; Smita Srivastava; Madhulika Singh; Yogeshwer Shukla
Journal: Journal of Toxicology
 (2009)

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## Abstract

Glyphosate (N-(phosphonomethyl) glycine,C3H8NO5P), a herbicide, used to control unwanted annual and perennial plants all over the world. Nevertheless, occupational and environmental exposure to pesticides can pose a threat to nontarget species including human beings. Therefore, in the present study, genotoxic effects of the herbicide glyphosate were analyzed by measuring chromosomal aberrations (CAs) and micronuclei (MN) in bone marrow cells of Swiss albino mice. A single dose of glyphosate was given intraperitoneally (i.p) to the animals at a concentration
of 25 and 50 mg/kg b.wt. Animals of positive control group were injected i.p. benzo(a)pyrene
(100 mg/kg b.wt., once only), whereas, animals of control (vehicle) group were injected i.p. dimethyl sulfoxide (0.2 mL). Animals from all the groups were sacrificed at sampling times of 24, 48, and 72 hours and their bone marrow was analyzed for cytogenetic and chromosomal damage. Glyphosate treatment significantly increases CAs and MN induction at both treatments and time compared with the vehicle control (P<.05). The cytotoxic effects of glyphosate were also evident, as observed by significant decrease in mitotic index (MI). The present results indicate that glyphosate is clastogenic and cytotoxic to mouse bone marrow.


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## Body

## 1. Introduction

Pesticides, including herbicides, insecticides, and
fungicides are used extensively to improve crop yields and as a
result, they accumulate in the environment and humans unavoidably exposed to them
[1]. Pesticides tend to be very reactive compounds that can form
covalent bonds with various nucleophilic centers of cellular biomolecules,
including DNA [2–4]. Because of their biological activity, the indiscriminate
use of pesticides may cause undesired effects to human health. For
instance, the induction of DNA damage can potentially lead to
adverse reproductive outcomes, the induction of cancer, and many
other chronic diseases [5–8]. Epidemiological studies demonstrated that
occupational exposure to some pesticides may be related to several kinds of
cancer, including leukemia [9], bladder [10], and pancreatic cancers [11].To assess the genetic damage induced by physical and
chemical agents including pesticides, various test systems have been described
in bacteria, in mammalian cells in vivo and in vitro and in plants [12–14].
Arguably, the most reliable genotoxicity evaluation for human health
risk is conducted in mammals by the induction of chromosomal aberrations (CAs)
and micronuclei (MN). In this regard, particular attention is focused on CAs
because these are considered as early warning signals for neoplastic
development [15, 16]. MN are defined as small, round, DNA containing
cytoplasmic bodies formed during cell division by loss of acentric chromatin
fragments and/or whole chromosomes and are used as a fast and reliable assay
for detecting clastogenic or aneugenic action [17]. CAs qualitatively and
quantitatively detect clastogenic activity, while the MN assay detects both clastogenic effects and damage to the mitotic apparatus, some of
which might have aneugenic consequences[18].Glyphosate [chemical name: N-(phosphonomethyl)glycine - isopropylamine (IPA) salt; C3H8NO5P;
Figure 1], commonly sold in the commercial formulation named Roundup, Rodeo, Touchdown,  and so forth, has been a frequently used herbicide on both cropland
and noncropland areas of the world since its introduction in the 1970s [19].
Roundup (CAS # 1071-83-6) is a liquid water soluble organophosphorus herbicide,
containing glyphosate as its active ingredient and surfactant (polyoxyethyleneamine)
that enhances the spreading of spray droplets when they contact foliage. As a
herbicide Roundup works by being absorbed into the plant not only through its
leaves but also through soft stalk tissue and applied at concentrations ranging
from 0.26–1.152% of active ingredient,  that is, glyphosate (20). Plants treated with
glyphosate slowly die over a period of days or weeks [20]. Glyphosate is
transported throughout the plant where it inhibits the shikimic acid pathway,
which participates
in the biosynthesis of phenylalanine and tyrosine and is also the major
pathway in the biosynthesis of most plant phenolics [21]. Because this specific biologic pathway operates
only in plants and microorganisms, the mechanism is not considered to be a risk
for humans. Nevertheless, genotoxic, hormonal, and enzymatic effects of
glyphosate in mammals have been reported [20, 22–25]. In rats, glyphosate was
found to decrease the activity of some detoxifying enzymes, cytochrome P-450,
and monooxygenase activities and the intestinal activity of aryl hydrocarbon
hydroxylase when injected into the abdomen [26].Figure 1
Chemical structure of glyphosate.Li and Long [27] reported nonmutagenic effects from glyphosate in Salmonella typhimurium, Escherichia
coli, Bacillus subtilis, Chinese hamster ovary cells gene mutation
assay and chromosomal aberration in rat bone marrow cells. However, some other studies stated that glyphosate
treatment on human lymphocytes in vitro resulted in increased sister chromatid exchanges [18, 22], CAs [22, 28], and oxidative
stress measured by glucose 6-phosphate dehydrogenase (G6PD, marker of changes
in the normal cell redox state) enzyme activity [22]. Roundup was associated
with increased DNA adducts in mice [23] and DNA damage in Rana catesbeiana tadpolesas assessed by using Comet
assay test [29]. Beside these, several
assays also have demonstrated genotoxic activities of roundup, such as
induction of reverse mutation in S. typhimurium (TA98 and TA100) and
sex-linked recessive lethal mutation in Drosophilamelanogaster [12, 28, 30] whereas glyphosate alone did not show these
effects. In mammalian cells glyphosate was not also mutagenic [19].
Thus, so far there have been conflicting reports on the genotoxic hazards
associated with the use of glyphosate.On the basis of the information available, U.S.
Environmental Protection Agency [31] and the World Health Organization [32] reviewed
the toxicology data on glyphosate and concluded that glyphosate is not
mutagenic or carcinogenic in humans. On the contrary, few recent studies have demonstrated
cytotoxic effects of glyphosate [22, 33, 34]. Considering the widespread and
frequent use of glyphosate throughout the world, ongoing risk assessment is of
importance. In the present study we reported the genotoxic potential of
glyphosate in mouse bone marrow cells.


## 2. Materials and Methods

### 2.1. Chemicals

Roundup containing active ingredient
glyphosate  >41% SL (IPA salt) was purchased from, Monsanto India Ltd.
(Mumbai, India). Benzo(a)pyrene [B(a)P], colchicine and Giemsa were obtained from Sigma Chemical Company (St. Louis, USA). The rest of the chemicals used in the
study were of analytical grade purity and obtained locally.


### 2.2. Animals and Treatment

Swiss albino mice (Male, 18   ±   2 g b.wt.; age: 10–12
weeks) were obtained from the Indian Institute of Toxicology Research (Lucknow,
India) animal breeding colony. The ethical approval for the experiment was
obtained from Institutional Ethical Committee. Animals were randomly selected
and housed in polycarbonate boxes with steel wire tops and rice husk bedding. They
were maintained in controlled atmosphere of 12 hours dark/light cycle,
25  ±  2C° temperature, and 57  ±  7% humidity with free access to pelleted feed (M/s.
Ashirwad, Chandigarh, India) and fresh tap water.The animals were divided into four groups of 15 animals
each in two sets. The animals of group I were used as a control group and intraperitonialy
(i.p.) treatment DMSO (0.2 mL, once only) was given. The animals of
group II were served as positive control and only B(a)P was given at the single
dose of 100 mg/kg b.wt. i.p. In groups III and IV single dose of glyphosate
(diluted appropriately in DMSO) was given i.p. at the dose of 25 and 50 mg/kg b.wt., respectively.


### 2.3. Chromosomal Aberration Assay

After completion of the treatment period 5 animals
from each group of set 1 were sacrificed at the sampling time of 24, 48, and 72 hours,
respectively, by cervical dislocation (colchicine was given at a dose of 4 mg/kg
of the b.wt. at 2 hours prior to sacrificing the animals to arrest cycling cells
in metaphase). Cytogenetic analysis was performed as per the protocol of
Preston et al. [35]. Briefly, the bone marrow was flushed out from both femurs
using Hanks buffered salt solution (pH 7.2). The cells were centrifuged at
1000 rpm for 5 minutes  and the pellet was redispersed in a hypotonic solution of
0.56% (w/v) KCl for 30 minutes  at 37C° to permit osmotic swelling of
cells. Swollen cells were fixed in ice-cold Carnoy’s fluid, dropped onto
slides, and stained with phosphate-buffered 5% Giemsa solution. A total of 75
well spread metaphase plates per animal in each group was analyzed for
chromosomal aberrations at a magnification of 100x and the mitotic index (MI)
was calculated from a scan of 2000 cells per animal. The chromosomal
aberrations were classified as breaks, fragments, and exchanges. The incidence
of aberrant cells was expressed as the percentage of damaged cells (aberrant
metaphases).Mitotic Index (MI) %:(1)Numberofdividingcells×100Totalnumberofbonemarrowcellscounted,Incidence
of aberrant cells (%): (2)Totalnumberofaberrantmetaphases×100Totalnumberofmetaphasescounted.


### 2.4. Micronuclei Induction Assay

The rest of 5 animals from each group of set 2 were
sacrificed after 24, 48, and 72 hours of treatment and the frequency of micronucleated
polychromatic erythrocytes (MNPCEs) was evaluated using a modified protocol of Schmid [36]. The bone
marrow was flushed from both femurs using Hanks’ buffered salt solution, 1%
(w/v) bovine serum albumin, and 0.15% (w/v) EDTA (pH 7.2). Evenly spread bone
marrow smears were stained by using the May-Grunwald and Giemsa protocol. A
minimum of 2000 erythrocytes was scored for each treated and control group. The
stained slides were scored for number of MNPCE’s/1000 PCE’s.


### 2.5. Statistical Analysis

The data was analyzed for mean values and standard
error (mean   ±   SE) for all groups. Statistical comparisons were made using
Studentst-test, and P < .05
was considered significant.


## 2.1. Chemicals

Roundup containing active ingredient
glyphosate  >41% SL (IPA salt) was purchased from, Monsanto India Ltd.
(Mumbai, India). Benzo(a)pyrene [B(a)P], colchicine and Giemsa were obtained from Sigma Chemical Company (St. Louis, USA). The rest of the chemicals used in the
study were of analytical grade purity and obtained locally.


## 2.2. Animals and Treatment

Swiss albino mice (Male, 18   ±   2 g b.wt.; age: 10–12
weeks) were obtained from the Indian Institute of Toxicology Research (Lucknow,
India) animal breeding colony. The ethical approval for the experiment was
obtained from Institutional Ethical Committee. Animals were randomly selected
and housed in polycarbonate boxes with steel wire tops and rice husk bedding. They
were maintained in controlled atmosphere of 12 hours dark/light cycle,
25  ±  2C° temperature, and 57  ±  7% humidity with free access to pelleted feed (M/s.
Ashirwad, Chandigarh, India) and fresh tap water.The animals were divided into four groups of 15 animals
each in two sets. The animals of group I were used as a control group and intraperitonialy
(i.p.) treatment DMSO (0.2 mL, once only) was given. The animals of
group II were served as positive control and only B(a)P was given at the single
dose of 100 mg/kg b.wt. i.p. In groups III and IV single dose of glyphosate
(diluted appropriately in DMSO) was given i.p. at the dose of 25 and 50 mg/kg b.wt., respectively.


## 2.3. Chromosomal Aberration Assay

After completion of the treatment period 5 animals
from each group of set 1 were sacrificed at the sampling time of 24, 48, and 72 hours,
respectively, by cervical dislocation (colchicine was given at a dose of 4 mg/kg
of the b.wt. at 2 hours prior to sacrificing the animals to arrest cycling cells
in metaphase). Cytogenetic analysis was performed as per the protocol of
Preston et al. [35]. Briefly, the bone marrow was flushed out from both femurs
using Hanks buffered salt solution (pH 7.2). The cells were centrifuged at
1000 rpm for 5 minutes  and the pellet was redispersed in a hypotonic solution of
0.56% (w/v) KCl for 30 minutes  at 37C° to permit osmotic swelling of
cells. Swollen cells were fixed in ice-cold Carnoy’s fluid, dropped onto
slides, and stained with phosphate-buffered 5% Giemsa solution. A total of 75
well spread metaphase plates per animal in each group was analyzed for
chromosomal aberrations at a magnification of 100x and the mitotic index (MI)
was calculated from a scan of 2000 cells per animal. The chromosomal
aberrations were classified as breaks, fragments, and exchanges. The incidence
of aberrant cells was expressed as the percentage of damaged cells (aberrant
metaphases).Mitotic Index (MI) %:(1)Numberofdividingcells×100Totalnumberofbonemarrowcellscounted,Incidence
of aberrant cells (%): (2)Totalnumberofaberrantmetaphases×100Totalnumberofmetaphasescounted.


## 2.4. Micronuclei Induction Assay

The rest of 5 animals from each group of set 2 were
sacrificed after 24, 48, and 72 hours of treatment and the frequency of micronucleated
polychromatic erythrocytes (MNPCEs) was evaluated using a modified protocol of Schmid [36]. The bone
marrow was flushed from both femurs using Hanks’ buffered salt solution, 1%
(w/v) bovine serum albumin, and 0.15% (w/v) EDTA (pH 7.2). Evenly spread bone
marrow smears were stained by using the May-Grunwald and Giemsa protocol. A
minimum of 2000 erythrocytes was scored for each treated and control group. The
stained slides were scored for number of MNPCE’s/1000 PCE’s.


## 2.5. Statistical Analysis

The data was analyzed for mean values and standard
error (mean   ±   SE) for all groups. Statistical comparisons were made using
Studentst-test, and P < .05
was considered significant.


## 3. Results

In the results of chromosomal aberration assay, the
percent incidence of aberrant cells in positive control B(a)P treated groups
were found to be 12.76, 14.35, and 15.22 in 24, 48, and 72 hours of sampling time,
respectively, in comparison to 1.88, 1.92, and 1.75 of untreated group I (Table1, Figure 2). The frequency of percentage aberrant cells was also found to be
significantly (P < .05) increased in glyphosate treated groups in dose- and
time-dependent manner. The frequency of percent aberrant cells in glyphosate
(25 mg/kg b.wt.) treated group III was found increased to 5.86, 7.24, and 7.76
in 24, 48, and 72 hours of sampling time, respectively, while in group IV (50 mg/kg b.wt.) it was 7.46, 8.85, and 9.24, respectively (Table 1, Figure 2).Table 1
Effect of glyphosate
treatment on induction of chromosomal aberration
in swiss albino
mice.
GroupsUntreatedB(a)PGlyphosateGlyphosate(100 mg/kg b.wt)(25 mg/kg b.wt)(50 mg/kg b.wt)Breaks0.36 ± 0.15.65 ± 0.42.86 ± 0.23.79 ± 0.16Fragments0.17 ± 0.011.59 ± 0.030.39 ± 0.11.94 ± 0.0224 hoursExchange0.26 ± 0.020.69 ± 0.20.47 ± 0.30.41 ± 0.01of treatmentMultiple
damage1.02 ± 0.074.83 ± 0.32.14 ± 0.41.32 ±  0.07Total
no. of1.81±0.0312.76±0.17*5.86   ±     0.12*7.46±0.14*aberrant cellsNumber ofBreaks0.33 ± 0.26.84 ± 0.53.37 ± 0.054.51 ± 0.07aberrant cellsFragments0.19 ± 0.012.63 ± 0.70.46 ± 0.030.89 ± 0.01(%) after48 hoursExchange0.23 ± 0.10.83 ± 0.030.59 ± 0.010.54 ± 0.02of treatmentMultiple
damage1.17 ± 0.044.05 ± 0.042.82 ± 0.062.91 ± 0.16Total
no. of1.92±0.0314.35±1.27*7.24±0.15*8.85±0.14*aberrant cellsBreaks0.34 ± 0.026.91 ± 0.104.42 ± 0.074.49 ± 0.13Fragments0.15 ± 0.012.93 ± 0.040.53 ± 0.020.82 ± 0.0272 hoursExchange0.19 ± 0.011.65 ± 0.060.47 ± 0.030.63 ± 0.02of treatmentMultiple
damage1.12 ± 0.043.73 ± 0.12.34 ± 0.093.30 ± 0.15Total
no. of1.80±.0515.22*   ±      1.197.76   ±
0.4*9.24±0.18*aberrant cellsMean
±    SE of animalsn = 5.P*<.05.Figure 2
Mutagenic
activity of glyphosate in Swiss albino mice showing incidence of aberrant cells
at sampling time of 24, 48, and 72 hours. Values
are expressed as mean   ±   SE of 5 animals. *Represent
significant increase over untreated control group at their respective sampling
time. Data are significant asP <  .05.Significant decrease in MI after B(a)P treatment was
noticed and evaluated as percentage of dividing cells which was found to be 2.46,
2.12, and 1.94 in group II in comparison to 4.88, 4.90, and 4.84 of untreated
control group I (Table2, Figure 2). A significant (P <  .05) decrease in MI was
also observed in glyphosate treated groups III and IV in comparison to untreated
controls (group I). Low-dose (25 mg/kg b.wt) glyphosate resulted in significant
decrease in MI by 4.12, 3.84, and 3.75 in 24, 48, and 72 hours of treatment while
high dose (50 mg/kg b.wt.) resulted in 3.54, 3.16, and 3.06, respectively (Table 2, Figure
3).Table 2
Effects of glyphosate treatment on mitotic index and micronuclei
induction in swiss albino mice.
Groups
(treatment)Mitotic index (MI) after
treatmentMicronuclei induction (MNPCEs/1000PCEs)
after treatment24 hours48 hours72 hours24 hours48 hours72 hoursGroup
I4.88±0.064.90±0.024.84±0.041.24±0.011.10±0.011.18±0.03(untreated)Group
II
B(a)P2.46±0.09#2.12±0.01#1.94±0.02#15.46±0.03*17.50±0.10*18.25±0.12*(100 mg/kg b.wt)Group
III
(glyphosate4.12±.05#3.84±0.04#3.75±0.03#3.87±0.02*5.76±0.08*6.12±0.07*dose 25 mg/kg b.wt)Group
IV
(glyphosate3.54±0.01#3.16±0.03#3.06±0.01#6.86±0.04*8.25±0.04*8.48±0.09*dose 50 mg/kg b.wt)Data shows mean    ±   SE of  5 animals in each
group.P#<.05 represents significant decrease
as compared to untreated control.P*<.05 represents significant increase as compared
to untreated control.MNPCEs: Micronucleated polychromatic
erythrocytes;PCEs: Polychromatic
erythrocytes.Figure 3
Cytotoxic
effects glyphosate in Swiss albino mice indicated by decrease in mitotic index
(MI) at 24, 48, and 72 hours of sampling time.
Values are expressed as mean   ±   SE of 5 animals. *Represent
significant decrease over untreated control group at their respective sampling
time. Data are significant asP  <  .05.The frequency of MNPCEs/1000PCEs in the present study was 15.46, 17.50, and 18.25 in 24, 48, and 72 hours of B(a)P treatment (group II) and
which was 1.24, 1.10, and 1.18 in control group I (Table2, Figure 3). Glyphosate
(25 mg/kg b.wt.) induced micronuclei induction in group III was 3.87, 5.76, and
6.12 whereas in group IV (50 mg/kg b.wt. glyphosate treated animals) it was 6.86,
8.25, and 8.48, in 24, 48, and 72 hours of
sampling period, respectively, (Table 2, Figure 4), suggesting the genotoxic
potential of glyphosate.Figure 4
Mutagenic
activity of glyphosate in Swiss albino mice showing increased micronuclei (MN) induction
at sampling time of 24, 48, and 72 hours. Values
are expressed as mean   ±   SE of five animals. *Represent
significant increase over untreated control group at their respective sampling
time. Data were significant asP  <  .05. MNPCEs: Micronucleated polychromatic erythrocytes and PCEs: polychromatic
erythrocytes.


## 4. Discussion

Results of the
present study reveals that single dose of glyphosate caused significant
incidence of chromosomal aberration and induction of micronuclei in a dose- and
time-dependent manner. Various cytogenetic results on commercial glyphosate are
problematic. They may depend on purity of the active agent and on the nature of
inert components. Surfactants and other inert compounds were previously
suggested to increase the toxicity of the herbicide [37]. In a recent study, Caiman
latirostris embryos were exposed at early embryonic stage to different
sublethal concentrations of Roundup (range from 50–1750 μg/egg), results from
both the comet assay and the MN test revealed a concentration dependent effect  [4].Glyphosate reported for
positive clastogenic and genotoxic effects in vitro [22, 27] which are consistent with our results (Tables 1 and
2). Chromosomal damage is considered to detect early effects of xenobiotic
insult and
evaluation of the frequency of CAs is a sensitive cytogenetic assay for
detecting exposure to mutagens and carcinogens [15]. In the present study, glyphosate induced CAs could be
attributed to early changes either an increase in induced DNA lesions or
interference with their repair (Table 1, Figure 1). Glyphosate has been
reported to cause DNA damage in erythrocytes of bullfrog tadpoles (R.
catesbeiana) [29]. However, few studies reported that glyphosate is weak or nonclastogenic in vivo [18, 28, 38].The MN induction assay was used as an additional
sensitive biological indicator of the damage to somatic cell genome of subjects
exposed to pesticide mixtures occupationally. It is known that the appearance
of MN is related to the loss of chromosome fragments due to chromosome breaks [39].
Our results revealed that there was elevation in the number of micronuclei in
the glyphosate exposed animals.  Because MN could be the consequence of the
mitotic spindle malfunction, it is possible that the glyphosate could also
express an aneugenic mode of action as inhibiting cell division and mitotic
spindle apparatus.The molecular mechanisms responsible for the
genotoxicity of glyphosate are not yet known clearly. However, the CAs and the
micronucleus formation observed in animals clearly indicate that these
compounds interact with chromatin DNA and induce damage there. Such
interactions/DNA damage may be caused by an increased incidence of alkali
labile sites in DNA as observed in kidney and liver with glyphosate treatment
in CD-1 mice [23]. Alkali labile sites are generally produced at abasic sites
in DNA and may be revealed under conditions that denature DNA secondary
structure. Peluso et al. [23] also reported a dramatic increase in the number
of oxidized guanine, 8-hydroxylguanine (8-OHdG), residues in DNA of liver cells
from mice treated with glyphosate which also may be the reason of chromosomal
damage in bone marrow cells of mice as observed in our study. It has also been shown
in our study that CAs and MN induction increases in time as well as dose-dependent manner. It could be due to the glyphosate induced toxicity which produces
reduced repair of spontaneous 8-OHdG and lead to an accumulation of
oxidation products [23].The sensitivities of two cytogenetic tests,
chromosome analysis and the micronucleus test, were compared by using mice
exposed to the substances glyphosate and B(a)P (Tables1 and
2). Both test
systems proved equally sensitive for genotoxicity assessment. Glyphosate at the tested doses significantly increased both the CAs rates and the MN
induction in comparison to control. Thus, our results indicate that glyphosate is able to induce CAs and MN accompanied by inhibition of
cell proliferation in Swiss albino mice following i.p. administration. In
view of the earlier reports on mutagenic activity of glyphosate in laboratory
experiments and from the present study, further studies are needed to assess
the possible health hazard from glyphosate.



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*Source: 308985-2009-04-14.xml*