Review of Literature

CHAPTER - 2
REVIEW OF LITERATURE

Pesticides are designed to kill and because their mode of action is not specific to one
species, they often kill or harm organisms other than pests, including humans. The WHO
estimates that there are 3 million cases of pesticide poisoning each year and up to
220,000 deaths, primarily in developing countries. The application of pesticides is often
not very precise. As the contribution of agro-chemicals towards increasing agricultural
production is well established, however, it may causes damage to the environment; the
ecosystem including the mankind. Pesticides are known to control insect pests, weeds,
diseases, rodents and pests in the storage. Though the pesticide industry in the developed
world has made good progress in the field of development and production of low risk/low
volume user and environment friendly pesticides formulation, pesticides in the
developing countries still now are mainly available in conventional formulations such as
dust, wettable powder, emulsifiable concentrates and solutions etc. The chemical
pesticides and fertilizers (agrochemicals) are commonly used in Indian agriculture. Farm
productivity is directly proportional to use of agrochemicals observed from the first green
revolution. Improper and unsafe use is quite common in India. Pesticides have been
considered potential chemical mutagens several experimental data has revealed that
various agrochemical ingredients possess mutagenic properties. The genotoxic potential
for agrochemical ingredients is generally low, as they yield positive results in few
genotoxicity tests. The lowest effective dose in single test is generally very high. Toxic
effect, mainly genotoxic potential is a primary risk factor for short and long-term effects
such as carcinogenic and reproductive toxicology.

However, most of the chemicals in use as pesticides are not completely selective.
The lack of highly selective pesticidal action represents a risk both for man and other
desirable forms of life present in the environment, thus concern has been raised about the
effects on the environment and human health caused by pesticide use, particularly in
those countries where surveillance, control and management activities are weak or poorly

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developed. Unlike other man-made chemicals, exposure to pesticides may affect a large
part of the human population, including workers and that part of the general population
who may experience exposure through domestic use, proximity to agricultural settings
and consumption of contaminated food. Moreover, a few subjects may be exposed to
pesticides through their use for public health purposes, as is the case for people living in
treated dwellings. However, exposure to pesticides primarily concerns the workers
involved in their industrial manufacture, formulation and application either in agriculture
or public health water (Table 2.1).

Table 2.1: Comparison between exposure patterns during production and use of
pesticides

Exposure in production Exposure in use

Duration of exposure Continuous and prolonged Variable and intermittent

Degree of exposure Quite stable Extremely variable

Type of exposure To one or few compounds To numerous compounds

either in sequence or
concomitantly

from use of mixtures

Skin absorption Easy to control Variable according to

work procedures

Ambient monitoring Useful Seldom informative

Biological monitoring Complementary to Very useful when

ambient monitoring available

Exposure in formulation shows features intermediate between agriculture and production

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In agricultural settings, exposure may also involve other agricultural workers
who enter treated fields (WHO, 1986b). Pesticides have long been considered potential
chemical mutagens (Vogel and Rohrborn, 1970; Fishbein et al., 1970; Hollaender, 1971).
Suspicion first arose regarding their mechanism of action. Indeed, their gene
mutagenicity has been verified in simple systems and even their injurious effects on
chromosomes were demonstrated in mammalian cells cultivated in vitro (Shaw, 1970;
Durham and Clara, 1972; Seiler, 1973). The mutagenic effects of pesticides were also
studied on human tissue culture cells (Dubinin et al., 1967; Chang and Klassen, 1968).
Recently the mutagenicity of some pesticides has also been proven in mammals in vivo
(Epstein et al., 1970; Dikshith, 1973). All of these studies have attracted attention
especially because pesticides, due to their extensive use, have considerable polluting
effects on the environment; thus several possibilities for human contamination also exist.
As pesticides are responsible for several adverse effects on human health other than acute
intoxications. Many studies have reported associations between exposure to agricultural
chemicals and various health outcomes, including different kinds of cancer (Daniels et
al., 1997; Khuder and Mutgi, 1997; Zahm and Ward, 1998) and degenerative diseases
(Engel et al., 2001; Gauthier et al., 2001; Jenner, 2001). Effects in immune,
hematological, nervous, endocrine and reproductive systems have been reported (Ojajarvi
et al., 2000; Ritz and Yu, 2000; Figa-Talamanca and Petrelli, 2001; Mourad, 2005), and
these compounds have been also associated with DNA damage in human populations
(Gomez-Arroyo et al., 2000; Undeger and Basaran, 2002; Costa et al., 2007; Ergene et
al., 2007; Muniz et al., 2008; Ali et al., 2008). Exposure to low-level of pesticides is
known to produce a variety of biochemical changes, some of which may be responsible
for the adverse biological effects reported in human and experimental studies (Gupta et
al., 1998; Banerjee et al., 1999; Panemangalore et al., 1999; Hernández et al., 2005).
Conversely, some biochemical alterations may not necessarily lead to clinically
recognizable symptoms, although all the biochemical responses can be used as markers
of exposure or effect (Panemangalore et al., 1999; López et al., 2007).

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CLASSIFICATION OF PESTICIDES

Pesticides may be classified in several ways. They may be classified according to the
target pests they destroy, for example, insecticides, herbicide, rodenticide and others they
may also be classified according to the chemical class they belong to for example
organochlorines, organophosphates, carbamates, pyrethriods, nitrophenols, nicotinoides
etc (David and Jeyaratnam 1996). Another system of classification may be according to
the degree or type of damage caused such as that developed by the World Health
Organisation. Other classification systems, based on combined functional and chemical
properties of the pesticides, have also been proposed (Hogstedt 1992).

PESTICIDES AND TOXICITY

Biological monitoring is the measurement of workplace agents or their metabolites either

in tissues, secreta, excreta, expired air or any combination of these, in order to evaluate
exposure and health risk compared to an appropriate reference (Berlin et al., 1984; WHO,
1986b). A presently unresolved problem is how to consider tests pointing out early
effects attributable to the exposure (Hoet, 1996). If these changes are ‘non adverse’ and
reversible, and if a dose–effect relationship is known, these indicators of sub-critical
effects could be used in the biological monitoring of exposure as markers of internal dose
rather than markers of potentially adverse effect. However, the distinction between early,
reversible health effects and adverse health effects, and hence between biological
monitoring of exposure and medical surveillance, is not always clear-cut. Biological
monitoring offers several advantages over ambient monitoring because a biological
parameter reflecting the internal dose is necessarily more closely related to the systemic
adverse effect(s) than any environmental measurement can be. Biological monitoring
provides an integrated estimate of exposure by all routes of absorption into the body
(respiratory, dermal and oral route) and evaluates the overall exposure as the sum of
different sources of contamination. It informs about long-term exposure in some cases
and helps to assess exposure of an individual subject as a result of individual movement
within the working environment and the individual factors influencing the

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pharmacokinetics of the exogenous substance (Maroni, 1983.). However, when routes of
exposure are integrated or combined, environmental monitoring could be helpful either to
clarify which route is more significant or to identify the compounds that have to be taken
into account in the biological monitoring practice. Regardless of the difficulties in
assessing risks of pesticide use on human health, the authorization for pesticide
commercialization in Europe currently requires data of potential negative effects of the
active substances on human health. These data are usually obtained from several tests
focused on e.g., metabolism patterns, acute toxicity, sub-chronic or sub-acute toxicity,
chronic toxicity, carcinogenicity, genotoxicity, teratogenicity, generation study, and also
irritancy trials using rat as a model mammal or in some cases dogs and rabbits
(Matthews, 2006). The respective toxicity tests for human health risk assessments
required by EPA (2009) are (1) the acute toxicity test, which assesses the effects of short-
term exposure to a single dose of pesticide (oral, dermal, and inhalation exposure, eye
irritation, skin irritation, skin sensitization, neurotoxicity), (2) the sub-chronic toxicity
test, which assesses the effects of intermediate repeated exposure (oral, dermal,
inhalation, nerve system damage) over a longer period of time (30–90 days), (3) the
chronic toxicity test, which assesses the effects of long-term repeated exposure lasting for
most of the test animal’s life span and intended to determine the effects of a pesticide
product after prolonged and repeated exposures (e.g., chronic non-cancer and cancer
effects), (4) the developmental and reproductive tests, which assess any potential effects
in the fetus of an exposed pregnant female (i.e., birth defects) and how pesticide exposure
may influence the ability of a test animal to reproduce successfully, (5) the mutagenicity
test which assesses the potential of a pesticide to affect the genetic components of the
cell, and (6) the hormone disruption test, which measures the pesticide potential to
disrupt the endocrine system (consists of a set of glands and the hormones they produce
that regulate the development, growth, reproduction, and behaviour of animals including
humans). The acute toxicity experiments are required for the calculation of the median
lethal dose (LD50), which is the pesticide dose that is required to kill half of the tested
animals when entering the body by a particular route. For example, if the substance is
swallowed the figure is an oral LD50, whereas if absorbed through the skin it is a dermal

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LD50. In addition, the acute inhalation lethal concentration (LC50), which is the
pesticide concentration required to kill half of the exposed (for 4 hours) tested animals to
a pesticide, is also calculated. Lethal concentration values are used when the route of
administration is by inhalation or intake via drinking water rather than oral or dermal etc.
These endpoints are used for WHO toxicity classifications of pesticides shown in Tables
2.2 and 2.3.

Table 2.2: Acute toxicity of pesticides according to WHO 2010 classification

Class Classification LD50 for the rat (mg/kg b.w.)

Oral Dermal

Solids Liquids Solids Liquids

Ia Extremely <5 <20 <10 <40

hazardous

Ib Highly 5–50 20–200 10–100 40–400

hazardous

II Moderately 50–500 200–2,000 100–1,000 400–4,000

hazardous

III Slightly >501 >2,001 >1,001 >4,001

hazardous

U Unlike to >2,000 >3,000 – –

present acute
hazard

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 Review of Literature

Table 2.3: Acute toxicity of pesticides according to the EPA classification

Acute toxicity to rat

Class Signal words Oral LD50 Dermal LD50 Inhalation

(mg/kg) (mg/kg) LC50 (mg/L)

I DANGER <50 <200 <0.2

II WARNING 50–500 200–2,000 0.2–2.0

III CAUTION 500–5000 2,000–20,000 2.0–20

IV CAUTION >5,000 >20,000 >20

(optional)

The oral LD50 is usually lower than the dermal LD50 since pesticides can enter the
bloodstream more easily through the stomach than through the skin (Nesheim et al,
2008). It must be noted that the LD50 values given in the WHO classification are for the
active ingredient, whereas these LD50 values must be modified to take account of the
concentration of the pesticide formulation actually used. This is because the actual
toxicity of a commercial pesticide product is significantly affected by the formulation.
For example, a highly toxic pesticide becomes more toxic when is formulated as
emulsifiable concentrate than as microcapsule suspension (Vasilakoglou and
Eleftherohorinos 1997). This is because the amount of the toxic active ingredient at the
time of application from the emulsifiable concentrate is much higher than that of the
microcapsule suspension. In addition, the emulsifiable concentrate is more toxic than the
microcapsule suspension because it includes very often toxic organic solvents (Surgan et
al, 2010). Also, the toxicity of the liquid formulation is usually much higher than that of

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the respective solid formulation since it is more difficult for a solid to pass through the
skin (Reifenrath, 2007).

Pesticides, on the basis of persistence in the environment, are of following types,

viz., organochlorines, neonicotinoids, organophosphates, carbamates and pyrethroids
which are discussed ahead supported by some concurrent researches done by the
scientists.

Organochlorine pesticides are considered persistent pesticides. These pesticides

have long environmental half-lives and tend to bio accumulate in humans and other
animals and thus biomagnify up to 70 000 times in the food chain (Borga et al., 2001).
Because migratory birds and other animals are at the top of the food chain, they carry
these persistent compounds with them wherever they go and are then transferred to the
very top of the food chain, humans (Rasmussen 1996). The contemporary nonpersistent
pesticides include organophosphates, carbamates, neonicotinoids, synthetic pyrethroids
and others. These pesticides have much shorter environmental half lives and tend not to
bioaccumulate. Organochlorine (OC) pesticides can be divided according to their
chemical structure into the following sub-groups: benzene hexachloride isomers, such as
lindane,. cyclodienes, such as aldrin, dieldrin, endrin, chlordane, heptachlor and
endosulfan,. DDT and analogues, such as methoxichlor, dicofol and chlorobenzylate.
Biological monitoring of OC exposure can be carried out through the determination of
intact pesticides or their metabolites in blood and urine. Because of their environmental
persistence, most OC pesticides have become ubiquitous pollutants; bioaccumulation
along the food chain results in detectable levels of these compounds in the highest animal
species, including man. In subsequent years, their use was discontinued in many
countries because of a persistent contamination of the environment.

Neonicotinoids are the most rapidly expanding insecticidal class since the launch
of the first compound, imidacloprid, by Bayer CropScience in 1991. In the 10 years that
followed, six additional neonicotinoid insecticides were launched: acetamiprid,
nitenpyram, thiamethoxam thiacloprid, clothianidin and dinotefuran.

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 Review of Literature

Structural diversity of neonicotinoid insecticides and year of market introduction Elbert

et al, 2008. Presently, neonicotinoid insecticides represent the most effective chemical
class for the control of sucking insect pests such as aphids, whiteflies, leaf- and plant
hoppers, thrips, some micro lepidoptera and a number of coleopteran pest species.
Furthermore, they constitute effective tools for controlling parasites of companion
animals/cattle and hygiene pests such as cockroaches, houseflies and termites. Among
neonicotinoids imidacloprid is an extensively used insecticide for crop protection in the
world wide from the last decade due to its low soil persistence and high insecticidal
activity at low application rate (Chao and Casida, 1997). It is fastest growing in sales as
insecticide globally because of its low selectivity for insects and apparent safety for
humans (Matsuda et al., 2001; Tomizawa and Casida, 2005). Its selective toxicity results
from its high affinity to insects nicotinic acetylcholine receptors compared to mammals
(Chao and Casida, 1997; Zhang et al., 2000; Tomizawa and Casida, 2003). In the present
study, imidacloprid (IM) is taken from the category of neonicotinoids.

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Organophosphorous (OP) compounds are derivatives of phosphoric and
thiophosphoric acids. Since these compounds act through the inhibition of
acetylcholinesterase (ACHE), the determination of ACHE activity in red blood cells
(RBC), and pseudocholinesterase (PCHE) activity in serum or plasma have been the most
reliable and widely used biological indicators of human exposure to OP insecticides. The
concern over the potential mutagenicity of organophosphates was increased when
trimethylphosphate, a simple organophosphate, was found to be mutagenic in mice. There
after the potential genetic effects of selected organophosphorus pesticides were
investigated using various methods in several laboratories and considerable information
has now accumulated for a preliminary and tentative estimation of the potential risk of
this group of insecticides for the human genome. A number of studies have been
conducted to investigate the mutagenic effects of organophosphate pesticides. Amer and
Aly (1992) studied the mutagenic effects of two organophosphorus insecticides,
tetrachlorvinphos (Gardona) and chlorpyriphos (Dursban), using chromosome aberrations
and SCE in cultured mouse spleen cells. They showed potent clastogenic effects. Degrave
and Moutschen (1984) observed a negative effect in mice treated with the
organophosphorus insecticide malathion. The genotoxic effect of methyl parathion,
another organophosphorus insecticide, has been studied in a number of test systems
where it was found to be both positive and negative. It induced significant MN in bone
marrow cells of rats and mice and sperm shape abnormalities in mice (Grover and Malhi,
1985; Mathew et al., 1990). However, in some of the studies methyl parathion was found
to be non-mutagenic or weakly mutagenic (Sobti et al., 1982; Kaur and Grover, 1983).
Bhunya and Behera (1984) reported the clastogenic effects of edifenphos in mouse bone
marrow cells. It showed karyotoxicity in embryonic shoot meristems in barley (Sharma
and Panneerselvam, 1990). However, it was found to be non-mutagenic in a bacterial test
system (Shirasu et al., 1976).

Carbamate insecticides are derivatives of carbamic acid, HOC(O)NH2. Carbaryl,

the first successful carbamate, was introduced in 1956. Two distinct qualities have made
it a widely used insecticide. First, it has very low mammalian, oral and dermal toxicity.

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 Review of Literature
Secondly, it has a rather broad spectrum of insect control. This has led to its wide use as a
lawn and garden insecticide. Propoxur is highly effective against cockroaches that have
developed resistance to organophosphates. It is commonly used by pest control operators
for the control of cockroaches and other household insects in restaurants, kitchens and
homes. Carbamates are used as either dusts or sprays. They may be absorbed through the
skin as well as by ingestion and inhalation. The immediate toxic effect of carbamates is
very similar to that of organophosphates, but the recovery is comparatively rapid.
Spontaneous recovery without medical treatment occurs generally within 4 hours of an
exposure which has produced symptoms and signs of headache, light-headedness or
dizziness, weakness, excessive salivation, nausea, or vomiting. More severe symptoms
and signs generally prompt medical treatment. Individuals have recovered from
poisoning that produced such symptoms and signs as visual disturbances, profuse
sweating, abdominal pain, incoordination, muscle fasciculations, breathing difficulties or
changes in the pulse rate. Carbamate (CBM) derivatives used as pesticides include CBM
insecticides, nematocides, fungicides and herbicides.

Synthetic pyrethroids are man-made chemicals that are produced to mimic the
effective action of natural pyrethrins. Their chemical structures are typically comprised of
a chrysanthemic acid analogue that is esterified most often with a ringed structure.
Pyrethroids are non-systemic pesticides that have contact and stomach action. Some
pyrethroids also have a slight repellent effect. In most formulations, piperonyl butoxide is
added as a synergist. In the past several years, the use of synthetic pyrethroids has
escalated as the use of the more toxic OP and carbamate insecticides has been curtailed.
Several reports evidenced that lambda-cyhalothrin exerted its neurotoxic effects through
voltage-dependent sodium channels (Soderlund et al., 2002; Ray and Fry, 2006) and
induced chromosomal aberrations, genotoxicity and micronucleus formation in rat bone
morrow cells (Campana et al., 1999; Fahmy and Abdallah, 2001; Celik et al., 2003,
2005). There are very few reports of imidacloprid and lambda cyhalothrin on mice which
are quoted along with other animals.

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 Review of Literature

CHROMOSOMAL ABERRATIONS AND MITOTIC INDEX

Chromosomal aberrations are induced via DNA breakage, their survival depends on the
fate of the DNA breaks. DNA breaks may either rejoin such that the chromosome is
restored to its original state, rejoin incorrectly or not rejoin at all. These last two cases
may be observable on microscopic preparations of metaphase cells. Study of various
mutagens by various workers is as below:

Obea et al. (2010) reported that DNA double-strand breaks (DSB) are known to
cause chromosomal aberrations (CA). The ends of DSB have different molecular
structures depending on the inducing agent. Restriction endonucleases, DNase I and
benzon nuclease produce DSB with 3’-OH and 5’-phosphate ends which should be easily
rejoinable (“clean” DSB). Other DSB-inducing agents, such as neocarzinostatin,
bleomycin and ionizing radiation induce DSB with chemically modified ends (“dirty”
DSB) which are not religatable before enzymatic pruning to make them “clean”. Both
“clean” and “dirty” DSB lead to CA whose quantities and distributions are quite similar.
That the number and not the molecular structures of DSB are essential for the production
of CA. All DSB-inducing agents induce exchange events in the G1 phase of the cell cycle
which look like sister chromatid exchanges (SCE) and are therefore called “false” SCE.
“False” SCE seem to be mainly intra changes and would therefore outweigh the
frequencies of interchanges. Consequently, the factor F of inter- to intrachanges would
then be <1, indicating that the majority of DSB would lead to CA inside the chromosome
domains in which they were induced and not between different chromosomes.

Fahmy and Abdalla (2001) studied single oral treatment with the different doses
of pyrethrins and lambda cyhalothrin and found significant increase in the percentage of
chromosomal aberrations in mouse bone-marrow. Concerning the different types of the
induced aberrations, gaps were found to be the most sensitive type induced after
treatment with the 2 pyrethroids. The aberrations were mainly of chromatid type.
Numerical chromosomal aberrations in the form of endomitosis (endomitotic
reduplication) were also reported in all treated groups.

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Degraeve and Moutschen (1984) reported no increase of the percentage of chromosome
aberration in both bone marrow cells and spermatogonia of mice treated with malathion
Soheir et al, 2002 found that cytogenetic effect of malathion residues in wheat grains
stored and feeding them for 4weeks had a non-significant effect with respect to the
induction of chromosomal aberrations or SCEs (sister chromatid exchanges). However
significant chromosome damage and increase of SCEs were observed in mice fed with
wheat grains stored for 12 weeks. The maximum effect was recorded in mice fed for 12
weeks with the grains treated with the highest tested dose and stored for 24 weeks. Giri et
al. (2002) while evaluating genotoxic effects of malathion reported that the metaphase
analysis of the bone marrow cells revealed various types of chromosomal aberrations
which consisted of chromatid and isochromatid types of gaps and breaks, double minutes
(included among isochromatid breaks), exchanges, and sister chromatid unions (SCU).
Chromatid type breaks were noted to be more frequent than others. Relatively higher
frequencies of gaps were observed for all the doses tested. A tentative assessment of the
distribution of breaks and gaps revealed that the distal regions of the long chromosomes
were more vulnerable to malathion. Malathion induced a significantly higher frequency
of aberrations for all the three doses tested at both the time points (24 and 48 h) as
compared to the control. Nada and Saleha, 2008 reported ethephon for its genotoxic
effect in both somatic (bone marrow) and germ (spermatocytes) cells of male mice at
three different dose levels and increase in chromosomal aberrations (structural and
numerical) was reported in most treatment with ethephon in both somatic and gametic
cells. In bone marrow cells, the significant structural chromosome aberrations were in the
form of chromatid gaps & breaks, deletions, fragments and centric fusion, while in
spermatocytes were in the form of chain, autosome univalent and x-y univalent. On the
other hand, the numerical aberrations, such as aneuploidy (hypoploidy 2n- and
hyperploidy 2n+), were below the significant level. Concerning the sperm shape
abnormalities, there was no significant increase over the control groups. The results were
attributed to the alkylating potency of the organophosphorus pesticide “herbicide” and
also to its cytotoxic effects. Their study concluded that ethephon has clastogenic effects.

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Giri et al. (2002) described the genotoxic effects of fenvalerate evaluating using
structural chromosome aberration (CA) and sister chromatid exchange (SCE) assays in
mice. Out of the three doses tested, statistically significant increase in CA was found
(following intra peritoneal treatment) in highest dose of fenvalerate for 24h and 48h only.
Neither the acute doses of low and medium dose, nor the sub-acute dose of fenvalerate
could induce any significant effect. The anti-genotoxic effect of apigenin (one of the
several active ingredients found naturally in many fruits and vegetables) against the
genotoxic damage induced by mitomycin C on mouse bone marrow cells using sister
chromatid exchanges (SCEs) and chromosomal aberrations was reported by Siddique and
Afzal (2009). For chromosomal aberration analysis the mean percent values of abnormal
metaphase were not significant at low, medium and high treatment of Apigenin. The
treatment of 2 mg/kg body weight of MMC induced (28.0±2.00)significant number of
abnormal metaphases as compared to normal (1.40±0.52) and significant dose dependent
decrease in number of abnormal metaphases was observed when low (20.4±1.80),
medium (18.4±1.73) and high (16.8±1.67) treatment of Apigenin were given along with
2 mg/kg body weight of MMC. Thus their study clearly reported the antigenotoxic
potential of apigenin against MMC on mice bone marrow cells. Hiroyuki Tateno (2008)
compared assisted reproductive technology, including in vitro fertilization (IVF) and intra
cytoplasmic sperm injection (ICSI), where incidences of structural chromosome
aberrations in embryos produced by ICSI with normal spermatozoa were considerably
high compared to those in embryos produced by conventional IVF technique. His study
suggested that chromosomal aberrations generated in ICSI one-cell embryos derived from
cauda epididymal spermatozoa after the incubation in PB1media for sperm incubation for
6 h were lethal. Singh et al. (2003) evaluated the genotoxicity of lomefloxacin, a
diflourinated antibacterial drug, by employing mouse in vivo chromosomal aberration
test in bone marrow cells and dominant lethal mutation assay in germ cells. Statistically
significant reduction in mitotic index (7%), increase in chromosomal aberrations /cell and
percent abnormal metaphase was observed only at the highest dose of the drug. In the
dominant lethal mutation assay, a statistically significant decrease in the number of
implants/female, compared to vehicle control, was noticed only in the females mated with

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males treated with 32 mg/kg b.w. during the third week of mating, while statistically
significant reduction in live implants/female was reported at low and medium doses
during the second and third weeks of mating. Nevertheless, no significant change in the
number of dead implants/female was reported after lomefloxacin treatment. Thus results
seems to indicate that lomefloxacin was a weak clastogen in the bone marrow cells and
non-mutagenic in the germ cells of mouse in vivo.

Genotoxicity of crotonaldehyde whose human exposure occurs from both man-

made and natural sources was reported by Jha et al. (2007). Crotonaldehyde has been
identified in exhaust from jets, automobiles and diesel engines, from tobacco smoke and
from the combustion of polymers and of wood. Treatment induced dose-dependent and
statistically significant decrease in mitotic index and increase in chromosome aberrations
per cell (CAs/cell excluding gaps and stickiness and pulverizations) and percent aberrant
metaphase (excluding gaps) in the bone marrow cells at 6, 12 and 24 h after treatment.
The overall result reports a positive dose–response relationship between treatment and
induction of chromosomal aberrations in the somatic and germ cells and dominant lethal
mutation in the germ cells. Ezzat (2002) described protective effect of calcium given
orally by gavage with two doses were evaluated against clastogenecity induced by lead
acetate with two concentrations on bone marrow and spermatocyte. The percentage of
chromosomal aberrations which induced with the two doses were statistically significant
after one, two and 7 days post treatment, recorded the highest values after 2 days. The
percentage of aberrations reduced after 7 days, but it still has a significant value. The
mean percentage of the recorded aberrant cells was significant even after excluding the
percentage of gaps cells of mice in vivo. As a result of orally gavage with calcium
chloride to mice treated with lead acetate, it recorded a retarded values of the aberrant
percentage. Choudhury et al. (1997) reported that the clastogenic affects of prolonged
exposure to sodium arsenite, a strong clastogen, were reduced by a highly significant
amount when crude garlic extract, in the dose used, was given daily to the mice by
intubation for the same period.

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The cytogenetic effect of orally treated 2,4-dichlorophenoxy acetic acid (2,4-D)
high dose induced significant percentage of aberrations where as two lower doses did not
affect the induction of chromosome aberrations significantly after single oral treatment
and its metabolite 2,4-dichlorophenol (2,4-DCP) intraperitoneally did not induce
significant percentage of aberrations in bone-marrow of mice as reported by Amer and
Fawzia (2001). Ritter and Durante (2010) reviewed the relative biological effectiveness
of heavy ions for the induction of cytogenetic damage which strongly dependent on the
time between irradiation and chromosome harvest, due to cell-cycle delays and loss of
heavily damaged cells. Heavy-ions induce a high fraction of complex-type exchanges,
and possibly unique chromosome rearrangements. Feng et al. (2009) analysed the
anticlastogenic effect of micrometer powder of selenium-enriched green tea (MSTP)
using a chromosomal aberration assay in mouse testicular cells. Results indicated that
MSTP showed a significant capability to reduce the incidence of Mitomycin–C induced
chromosomal aberrations in spermatocytes from 22.7% to 6.7%. Mukherjee and
Chakrabarti (1997) while evaluating in vivo genotoxic and clastogenic potentials of
Acesulfame-K, a sweetening agent in swiss albino male mice found a positive response
suggesting that Acesulfame-K is capable of interacting with DNA to produce genetic
damage.

CHROMOSOMAL ABERRATIONS AND MICRONUCLEUS

Micronuclei frequencies have been considered to be a reliable index for detecting

chromosome breakages and loss. For genetic toxicology studies, the formation of
micronucleus in bone marrow and peripheral blood erythrocytes is recognised as one of
the established methods for in vivo cytogenetic assays. Bone marrow cytogenetics is also
a useful shortterm technique, for elucidating the mechanisms involved for the clastogenic
and anticlastogenic activity of substances

Aydemir and Bilaloglu (2003) studied the genotoxic effects of gemcitabine and
topotecan in mouse bone marrow cells using the micronucleus and chromosomal
aberration test systems. Gemcitabine increased the frequency of micronuclei, particularly

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at the median dose for the 24-, 36-, and 48-h sampling intervals. It had cytotoxic effects
on the bone marrow and decreased the polychromatic/normochromatic erythrocyte ratio
dose-dependently for all sampling intervals. Gemcitabine significantly decreased the
mitotic index at the 24-h time point. It increased the number of abnormal cells and
induced a significant increase in total chromosomal aberrations. For the 6-h sampling
time, gemcitabine neither induced chromosomal aberrations nor reduced the mitotic
index. Topotecan also induced high levels of micronuclei, particularly for the 24- and 36-
h sampling times and it decreased the polychromatic/normochromatic erythrocyte ratio
for all sampling intervals, which is indicative of bone marrow cytotoxicity. The bone
marrow metaphase analysis showed that topotecan significantly elevated the number of
abnormal metaphases and total chromosomal aberrations at 6 and 24 h, in a dose-
dependent manner. It also decreased the mitotic index for both sampling intervals. Thus,
the results of this study indicate that the two chemotherapeutics gemcitabine and
topotecan have cytotoxic and genotoxic effects in mouse bone marrow. Naik and
Vijayalaxmi (2009) concluded that Bisphenol-A (BP-A) is a xenobiotic estrogenic
compound which is reported to be non-significant and failed to induce conventional
chromosomal aberrations and micronuclei in swiss albino mice.

Abbes et al. (2007) conducted a study to evaluate the ability of hydrated sodium
calcium aluminosilicate (HSCAS) to protect Balb/c mice against cytotoxicity and
genotoxicity induced by ZEN (Zearalenone, a potent estrogenic metabolite). The results
were that ZEN was cytotoxic and genotoxic to Balb/c mice, as indicated by the increase
in the frequencies of micronucleated polychromatic erythrocytes (PCEMN) and of
chromosomal aberrations in bonemarrow cells. The simultaneous intra-gastric
administration of HSCAS with ZEN resulted in a reduction in the number of PCEMN and
a decrease of the chromosomal aberration frequency, and an increase in the number of
polychromatic erythrocytes (PCE) in bone-marrow cells, compared with those in the
group treated with ZEN alone. Concluding that HSCAS itself was safe and efficient in
the prevention of the toxic effects of ZEN in the gastrointestinal tract. Surjyo et al. (2005)
demonstrated that the chronic feeding of p-dimethylaminoazobenzene (p-DAB) produced

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increased numbers of chromosome aberrations, nuclear anomalies and sperm-head
abnormalities, as compared to normal untreated controls, generally in a time-dependent
manner until 60 days, after which the anomalies persisted, but rather erratically. I.D.
Adler et al. (1988) obtained positive results in the chromosomal bone marrow test and the
micronucleus assay with Acrylamide, but the effect increased linearly with dose.
Farghaly (2010) evaluated the chemo protective efficacy of Phytic Acid (PA) against
Acrylamide (AA) induced genotoxicity and biochemical disturbance in mice in vivo and
reported the protective role of phytic-acid against the genotoxicity and biochemical
disturbance of AA.

Both normal metaphase spreads and spreads with various types of chromosome
aberrations (CA) in the mice-fed with Phenobarbital (PB) a widely used antiepileptic
drug were reported by Biswas et al. (2004). The mitotic indices of bone-marrow cells
were slightly increased in the PB-fed mice as compared with controls that received
distilled water. Similarly, an apparent increase in the occurrence of both polychromatic
and normochromatic erythrocytes was noted. Palo et al. (2009) reported that Cytosine
Arabinoside (Ara-C), a antineoplastic drug induced statistically significant and dose
dependent increase in the percentages of aberrant metaphases and chromosomal
aberrations (CAs) at 24 h post-treatment, and micronuclei (MN) in polychromatic
erythrocytes (PCEs) in mice. However, there was no significant change in the mitotic
index (MI) at 24 h post-treatment, when compared to that of the control mice. Naya et al.
(2011) evaluated in vitro chromosomal aberration test, and an in vivo mouse bone
marrow micronucleus test for genotoxic potential of a high purity sample of single-wall
carbon nanotubes (SWCNTs). The chromosomal aberration test did not increase the
number of structural or numerical chromosomal aberrations, whether the test was
conducted with or without metabolic activation. In the in vivo bone marrow micronucleus
test also did not affect the proportions of immature and total erythrocytes, nor did it
increase the number of micronuclei in the immature erythrocytes of mice. Chauhan et al.
(2000) tested Carbofuran in in vivo cytogenetic effects in mouse bone marrow cells. Mice
exposed to carbofuran showed mild symptoms of toxicity such as tremors, lacrimation

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and inhibited MI in a dose-dependent manner. The frequency of chromosomal aberrations
increased with increasing doses and duration. Single oral exposure of carbofuran-induced
chromosome- type aberrations (fragments and ring formation, chromatid-type
aberrations) chromatid breaks and gaps and occasionally pulverisation in mouse bone
marrow cells. The frequency of micronucleated cells was also found to be considerably
high in the exposed mice. Tanaka et al. (2008) detected chromosome aberration rate in
mouse splenocytes after long-term exposure to low-dose-rate (LDR) ɤ -rays increased
incidence of dicentrics and centric rings, this trend was also observed for the incidences
of micronuclei and trisomies of chromosomes.

MICRONUCLEUS

Genotoxic potential of jet fuels was investigated in mice treated dermally with either a
single or multiple applications of jet fuels and bone marrow smears were prepared to
examine the incidence of micronuclei (MN) in polychromatic erythrocytes (PCEs). No
statistically significant increase in the incidence of MN was observed in mice treated with
JP-8 or Jet-A when compared with those of untreated control animals. Percentages of
PCEs in the blood and bone marrow tissues of treatment group of mice were significantly
different from those in concurrent untreated control animals as repoted by Vijaylaxmi et
al. (2006). Ziemann et al. (2010) studied genotoxic potential of SO2 which is a
ubiquitous environmental pollutant and an important chemical intermediate in several
industrial processes. In vivo clastogenicity of SO2 did not induce micronuclei in
polychromatic erythrocytes of the bone marrow. Male and female animals showed a
highly significant increase in the frequency of micro nucleated PCE 24 h after CP (60
mg/kg b.w.) administration. Micronucleus frequencies amounted to 3.53% for male and
2.47% for female mice. Rajaguru et al. (1999) investigated the clastogenicity of the azo
dye using the murine bone marrow micronucleus Assay. They studied that he route of
administration had a significant effect on the frequency of micronucleus formation:
intraperitoneal injection was approximately two-fold less clastogenic than the equivalent
dose delivered orally. Mice bone marrow exposed to different doses of DR2 azo dye
resulted in significant increase in MPE at all concentrations, relative to vehicle only

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treated controls. PCE/NCE ratio showed a pronounced cytotoxic effect of the dye DR2 on
the bone marrow at doses higher than 4 mg/kg body weight. Musk ketone was evaluated
in an in vivo mouse micronucleus assay treated with 250, 500 or 1000 mg Musk
ketone/kg body weight by a single intraperitoneal injection in corn oil showed that under
the conditions of this test evaluated at 24, 48 and 72 h after dosing, Musk ketone did not
induce a significant increase in micro nucleated polychromatic erythrocytes in either
male or female mice at any dose or any time period as reported by Api and Gudi (1999).
Melatonin was tested using micronucleated polychromatic erythrocytes as an index of
damage in both bone marrow and peripheral blood cells of mice. The number of the
micronucleated polychromatic erythrocytes increased after paraquat administration both
in peripheral blood and bone marrow cells. The induction of micronuclei was time-
dependent with peak values occurring at 24 and 48 h. Mitomycin C, which was used as a
positive control, also caused the expected large rises in micronuclei in both bone marrow
and peripheral blood cells at 24, 48 and 72 h after its administration. increased the
number of MN-PCE at 24, 48, and 72 h, while no differences were observed in the
PCE/NCE ratio. Melatonin inhibited the increase in MN-PCE by more than 50% at 48
and 72 h. The ratio of PCE/NCE did not change after paraquat or mitomycin treatment as
reported by Ortiz et al. (2000). Andrew et al. (2000) studied atrazine, simazine, and
cyanazine which are widely used herbicides. None of the triazines investigated induced
MN in the bone marrow, even at doses that caused significant bone marrow suppression
and/or death. It was reported that atrazine, simazine, and cyanazine are not genotoxic as
measured by the bone marrow MN assay in mice following high dose exposures. Witt et
al. (2000) investigated tetrachloroazobenzene and tetrachloroazoxybenzene for toxicity.
Both chemicals reported significant increases in MN-NCE in male and female mice. In
contrast to these positive results in subchronic exposure studies, no significant increases
were seen in acute bone marrow MN tests in male mice administered three daily
injections of 50–200 mg/kg/day TCAB and TCAOB. In contrast to the positive results
observed in the 13-week peripheral blood micronucleated NCE tests, neither TCAB nor
TCAOB induced significant increases in MN in bone marrow PCE of male mice treated
by i.p. injection (50–200 mg/kg) three times at 24 h intervals. Bisphenol A (BPA) is a

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synthetic monomer widely used to polymerize polycarbonate plastics and resins. It is
shown in vitro to interfere with microtubules, producing aberrations in mitotic and
meiotic spindles. According to Pacchierotti et al. (2008), two daily oral BPA doses did
not induce any increase of micronucleus frequencies in polychromatic erythrocytes of
mouse bone marrow. Tripathi et al. (2008) evaluated that MN frequencies in the
reticulocytes and normochromatic erythrocytes of newborn mice after exposure of tested
chemicals reported significant increase in the micronucleated reticulocytes (MNRETs)
and micronucleated normochromatic erythrocytes(MNNCEs) in peripheral blood. There
was a significant increase in all target organs (liver, kidney, bone marrow and
lymphocytes) in all treated groups. Poca et al. (2008) reported that Malaria modulate the
activity of cytochrome-P450 enzymes involved both in the activation and detoxication of
xenobiotics, The background incidence of polychromatic erythrocytes with micronuclei
(MN-PCE) in malaria-infected mice was approximately twofold the background
incidence in non-infected controls. In non-infected mice, CPA, in doses up to 50 mg/kg
body weight, induced a dose-dependent increase in the frequency of micronuclei in
polychromatic erythrocytes (MNPCE) in the absence of any statistically detectable
reduction of PCE/NCE ratio. Frequency of micronuclei induced by model mutagens,
cyclophosphamide (CP), mitomycin-C (MMC) and bleomycin (BLM) hydrochloride
were tested using mouse bone marrow and reported to be effective in reducing MN
frequency, highest by MMC as reported by Vijaylaxmi and Venu (1999). Mengs et al.
(1997) reported the potential of emodin to induce micronuclei in polychromatic
erythrocytes (PCEs). Further, the mean number of NCEs was not substantially increased
as compared to the mean values of NCEs of the negative controls, indicating that emodin
had no cytotoxic effects on bone marrow cells. Studies reported that doses of
Ethylmethanesulphonate (EMS) up to 80 mg/(kg day) did not induce micronuclei in
mouse bone marrow. Only at higher dose levels the genotoxic activity of EMS became
apparent. Only at higher doses a clear increase was seen, with saturation of the effect at
the clearly cytotoxic dose of 260 mg/(kg day) by Gocke and Muller (2009). Benzene, a
ubiquitous pollutant, has been identified as a human leukemogen. It is known that
genotoxic agents can increase the frequency of DNA double-strand breaks (DSBs), which

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can initiate DNA recombinational repair mechanisms. Acute exposure reported
statistically significant and increase in the percentage of micro nucleated cells in adult
male bone marrow cells and in fetal liver and post-natal day 9 bone marrow cells of mice
exposed in utero. In dams, exposure to 400 mg/kg benzene resulted in a statistically
significant increase in the percentage of micronucleated bone marrowcells on gestational
day 16 compared to vehicle controls (mean±SD; 0.330±0.141 vs. 0.024±0.028 vehicle
group, p < 0.05) by Lau et al. (2009).

Herbold et al. (2001) studied ciprofloxacin which is widely used in antimicrobial

therapy and reported in vivo genotoxic activity using the micronucleus test in bone
marrow of mice, cytogenetic chromosome analysis in Chinese hamster and dominant
lethal assay in male mice. All in vivo genotoxicity revealed no genotoxic effect for
ciprofloxacin. In addition, ciprofloxacin was found to be non-carcinogenic in two rodent
long-term bioassays. No significant increase of the numbers of micro-nucleated
polychromatic or micro nucleated normochromatic erythrocytes was reported in the first
test 24, 48 or 72 h after treatment with 4000 mg/kg. The frequency of MN-PCE reported
metformin at 62.5, 125 and 250 mg/kg day and were found to be in the same range at 30
or 24, 48 and 72 h after treatment. The PCE/NCE ratio was in the same range except the
high dose of metformin at 48 and 72 h where the values were slightly less by Aleisa et al.
(2007). Attia (2007) investigated the potential of nicotine to induce micro-nucleated
polychromatic erythrocytes (MNPCE) in bone marrow of male and female mice with
evident signs of acute toxicity shown by the animals, mainly at the 8 and 16 mg/kg doses
of nicotine and the reduction in the % PCE. A sex dependent response was reported as
males having more MNPCE than females after treatment with 8 or 16 mg/kg nicotine and
sampling at 30 h. However, at 36 h more MNPCE were induced in females than in males,
suggesting different degrees of dose interaction in the sexes under the conditions of the
assay. The % MNPCE in the 10 vehicle-treated groups in the five experiments was within
the accepted spontaneous range for negative controls. A statistically significant increase
in the incidence of MNPCE over the control value was reported. Lewinska et al. (2007)
reported the genotoxic interactions between the common environmental pollutants:

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 Review of Literature
arsenic (As), cadmium (Cd) and benzo(a)pyrene (BaP), which are known to be human
carcinogens with BaP inducing a statistically significant increase in micronucleus (MN)
frequency at 48 h after administration. A statistically higher MN frequency was found in
bone marrow of animals exposed to As (0.92±0.29%) versus (0.38±0.13%, respectively.
In animals co-exposed to Cd (100 mg Cd2+/L) and BaP (200 mg/kg bw), the level of
MN in erythrocytes at the respective time-points studied did not differ from the level
observed in animals given BaP alone. Statistically significant effect was observed 48 h
after dosage. They were unable to found significant differences between the exposed and
control animals with respect to the ratio of polychromatic erythrocytes (PCE) per total
number of poly- and normo-chromatic erythrocytes (PCE + NCE). In vivo studies on
bone marrow polychromatic erythrocytes (PCE) from mice treated with Urografina with
purified sodium amidotrizoate and meglumine amidotrizoate separately or in combination
at the same ratio and concentration showed significant increase in the frequencies of
micronucleated polychromatic erythrocytes (MNPCEs) in both male (p = 0.0082 and p =
0.0062) and female (p = 0.0350 and p = 0.0101) mice treated with doses of 14.3 and 20.0
ml/kg body weight. Mann–Whitney test showed no significant differences in the
percentage of PCEs or in the frequencies of micronucleated polychromatic erythrocytes
(MNPCEs) between males and females of similarly treated groups as reported by
Deimlinga et al. (2009). Rosmarinic acid (RA) is a natural phenolic compound which
presents different biological activities such as antitumor, antibacterial, anti-inflammatory,
hepatoprotective and cardioprotective properties. Furtado et al. (2008) evaluated the
mutagenic and/or antimutagenic potential of rosmarinic acid on peripheral blood cells of
Swiss mice using the micronucleus assay. Which showed no increase in the frequency of
micronuclei in animals treated with different concentrations of RA when compared to the
negative controls. No significant difference in the induction of micronuclei was observed
between the groups treated with the different doses of RA and the negative control.

1,4-Dioxane, a widely used industrial chemical and rodent hepatocarcinogen,

reported to have produced mixed, largely negative results in the mouse erythrocyte
micronucleus assay. It was determined that 80–90% of the micronuclei in both tissues

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 Review of Literature
originated from chromosomal breakage. Small increases in centromere containing
micronuclei were also seen in the hepatocytes. Decreases in hepatocyte proliferation as
well as in the ratio of bone marrow PCE:NCE were also reported with significant
increase in the frequency of micro nucleated erythrocytes by Roy et al. (2005). The high
iron diet reported increased frequency of micronucleated polychromatic erythrocytes
(MnPCEs) as compared to low iron. Ascorbic acid supplementation in the low iron diet
did not show any effect on incidence of MnPCEs and protected against the increased
frequency of MnPCEs induced by the high iron diet. The frequency of micronuclei in the
bone marrow cells of mice treated with cyclophosphamide was significantly higher (P <
0.001) (Premkumar and Bowlus, 2003). Ximena et al., 2007 reported that there was no
significant difference (p > 0.05) between the sexes regarding the micronucleus frequency
in either the experimental or the control group. When the Mn frequencies of the three
strains were compared, the results for the CFW and BALB/c J mice strains did not differ
statistically (p > 0.05) for either the experimental or control groups but there were
significant (p < 0.05) differences between the CFW and NIH strains and the NIH and
BALB/cJ strains for the experimental and control groups, with the NIH strain always
showing the highest micronucleus frequency. Santos-Mello et al. (2001) described the
effects of the concentration and route of administration of non-radioactive cesium
chloride (CsCl) in inducing micronuclei in mouse bone marrow polychromatic
erythrocytes (PCEs). When the dose of 500 mg/kg body weight was administered per
orally, no significant incidence of micronuclei was reported. However, when the same
dose was administered intraperitoneally, a significant induction of micronuclei in PCEs
was reported. An increase in the frequency of MNPCEs was reported in all groups
treated with single metal compounds when compared with controls, for both treatment
periods (5 and 10 doses). Cadmium (Cd) treated groups showed always the highest
frequency of MNPCEs. Animals exposed to a simultaneous administration of two metal
compounds showed significant increases in the MNPCE frequencies when compared with
the respective control groups as reported by Tapisso et al. (2009). Increase in MNPCE
frequency at the 48 h sampling in animals receiving the 200 mg/kg dose reported in mice
bone marrow. A very marginal increase in mean and individual MNPCE values seemed

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to be apparent at the 72 h sampling in animals who received the 400 mg/kg dose. The
marked decrease in mean PCE/NCE ratio observed at the 72 h sampling in animals
receiving 400 mg/kg was considered indicative of bone-marrow toxicity by Guzman et
al. (2004).

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