Environmental Toxicology and Pharmacology 63 (2018) 103–114

Contents lists available at ScienceDirect

Environmental Toxicology and Pharmacology

journal homepage: www.elsevier.com/locate/etap

Review

Hazardous effects of chemical pesticides on human health–Cancer and other T

associated disorders
⁎
Akash Sabarwala,b, Kunal Kumara, Rana P. Singha,b,
a
School of Life Sciences, Central University of Gujarat, Gandhinagar, Gujarat, India
b
Cancer Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India

A R T I C LE I N FO A B S T R A C T

Keywords: Poisoning from pesticides is a global public health problem and accounts for nearly 300,000 deaths worldwide
Pesticides every year. Exposure to pesticides is inevitable; there are different modes through which humans get exposed to
Cancer pesticides. The mode of exposure is an important factor as it also signifies the concentration of pesticides ex-
Epigenetics posure. Pesticides are used extensively in agricultural and domestic settings. These chemicals are believed to
Oxidative stress
cause many disorders in humans and wildlife. Research from past few decades has tried to answer the associated
mechanism of action of pesticides in conjunction with their harmful effects. This perspective considers the past
and present research in the field of pesticides and associated disorders. We have reviewed the most common
diseases including cancer which are associated with pesticides. Pesticides have shown to be involved in the
pathogenesis of Parkinson’s and Alzheimer’s diseases as well as various disorders of the respiratory and re-
productive tracts. Oxidative stress caused by pesticides is an important mechanism through which many of the
pesticides exert their harmful effects. Oxidative stress is known to cause DNA damage which in turn may cause
malignancies and other disorders. Many pesticides have shown to modulate the gene expression at the level of
non-coding RNAs, histone deacetylases, DNA methylation patterns suggesting their role in epigenetics.

1. Introduction
Pests are any animals, plants or microorganisms that compromises
our food, health or comfort. Environmental Protection Agency (EPA)
describes pesticide as any substance which is used to prevent, destroy,
repel or mitigate any pest. According to USEPA report, the global ex-
penditures at the producer level were 56 billion dollars in 2012.
Between the years 2008–2012 herbicides accounted for maximum ex-
penditures in all years (45%) followed by expenditures on insecticides,
fungicides, and other pesticides. In 2012, U.S. expenditures accounted
for 21% of world expenditures on herbicides (including plant growth
regulators [PGRs]), 14% on insecticides, 10% on fungicides, and 23%
on fumigants.
Pesticides are used from our bedrooms to agriculture fields and the
later consume a substantial portion of total pesticides produced. In
terms of amount, maximum usage has been recorded for herbicides
followed by insecticides. Rachel Carson’s book “Silent Spring” opened a
new horizon of debate on the use of pesticides and destruction of non-
targeted flora & fauna and inspired the foundation of USEPA in 1970.
This debate ultimately affected the pesticide industries directly, hence
agrochemical industries spent thousands of dollars to contend the
message of her book (Dunn, 2012). In the twentieth century, pesticides
which were banned in the United States of America or never been re-
gistered under EPA were being exported to developing countries. The
workers employed in such industries were at a higher risk, and devel-
oping countries which were importing these chemicals have exposed
their greater populations. It was estimated that pesticide poisoning was
thirteen times higher in developing countries as compared to United
States (Swaminathan, 1982).
There are different ways in which humans get exposed to pesticides;
these exposures also differ in their intensities, which add up to their
differential effects. Workers involved in pesticide industries, transpor-
tation of these hazardous chemicals, farmers, fruit and vegetable sellers
and consumers get exposed to different concentrations of pesticides.
Exposure to pesticides is thought to be linked to numerous health dis-
orders such as Hodgkin's disease (HD), non-Hodgkin lymphoma (NHL)
(Luo et al., 2016; Wiklund et al., 1987), Parkinson’s disease (Brouwer
et al., 2017; Wang et al., 2014), endocrine disruption (Freire et al.,
2013; Mazur et al., 2015), respiratory and reproductive disorders
(Kirkhorn and Schenker, 2002). Pesticides are also believed to cause
cancers in humans, e.g. glyphosate is associated with breast cancer
(Thongprakaisang et al., 2013). The pesticides which contain alkyl

⁎
Corresponding author at: School of Life Sciences, Jawaharlal Nehru University, 104 Cancer Biology Laboratory, New Delhi-110067, India.
E-mail address: rana_singh@mail.jnu.ac.in (R.P. Singh).

https://doi.org/10.1016/j.etap.2018.08.018
Received 14 January 2018; Received in revised form 21 July 2018; Accepted 27 August 2018
Available online 01 September 2018
1382-6689/ © 2018 Elsevier B.V. All rights reserved.
A. Sabarwal et al. Environmental Toxicology and Pharmacology 63 (2018) 103–114

ureas and amines are found to be associated with brain tumors

(Musicco et al., 1988). The prostate cancer risk was found to increase in
people exposed to Agent Orange [a mixture of 2,4-di-
chlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid
(2,4,5-T), picloram, and cacodylic acid], which was heavily used by the
USA in Vietnam war (Ansbaugh et al., 2013). Dieldrin causes tumors of
lung, liver, lymphoid tissue, uterus, thyroid, mammary gland in test
animals at doses as low as 0.1 ppm (Norman, 1974). Several pesticides
are recognized as endocrine disruptor compounds (EDCs) which were
first recognized by the WHO in 2002. These are exogenous substances,
natural or synthetic, that interfere with endocrine systems and there-
fore may cause adverse effects on physiological processes including
development, growth, and reproduction in organisms and/or in their
progeny. Transcriptional activity of nuclear receptors is one of the
prime targets of EDCs, and several pesticides including organo-
chlorines, diphenyl ethers, organophosphorus pesticides, pyrethroids,
carbamates, acid amides, ureas were found to act as agonists of these
nuclear receptors (Kojima et al., 2011).
Oxidative stress is one of the major mechanisms which is associated
with several diseases and aging. Naturally, this phenomenon increases
with age and affects several vital processes in the body. Many pesticides
(paraquat, rotenone and maneb) cause Reactive Oxygen Species (ROS)-
mediated stress and neurodegenerative diseases. For instance, at cel-
lular level, Parkinson’s disease (PD) is closely associated with excessive Fig. 1. Estimated population groups at risk of pesticide exposure. Percent va-
production of ROS from mitochondrial respiratory complexes, which in lues of actual pesticide exposure cases via intentional & occupational, occupa-
turn causes serious injuries to mitochondrial DNA and other macro- tional and non-occupational modes.
molecules (Blesa et al., 2015; Nandipati and Litvan, 2016). Similarly,
Alzheimer's disease (AD), one of the most common disability in aged
intentional (accidental/suicidal), occupational and non-occupational
people, is characterized by abnormal deposition of amyloid β (Aβ)
exposures. Which may include long-term, short-term and in combina-
protein is also closely coupled with oxidative stress (Huang et al.,
tions with low and high doses (Davies et al., 1980). Davies and col-
2016). Long-term exposures at low doses of paraquat, dieldrin, orga-
leagues explained the risk to population groups of pesticides (Fig. 1)
nochlorine, and organophosphates have found to generate ROS-medi-
(Davies et al., 1980).
ated neurotoxicity which is positively related to Alzheimer's disease
(Yan et al., 2016).
Though there are several associated threats, still the necessity of
2.1. Intentional exposure
these pesticides forces us to use these substances regularly. These
chemicals also support us to fight against deadly diseases and ensure
Pesticides rank third in the world as a source to attempt suicides in
the availability of food supply to the exponentially growing human
most developing countries. Its easy availability is one of the reasons for
population at affordable prices. In a hypothetical condition of banning
its choice, especially in rural areas. An estimated 300,000 deaths occur
pesticides in the USA alone, it was estimated that supplies of food grains
alone in China and South East Asian countries due to intentional in-
such as wheat, corn and soybeans would drop by 27% (Knutson et al.,
gestion of pesticides (Bertolote et al., 2006; Gunnell and Eddleston,
1990).
2003). To evaluate the intentional and accidental exposure to these
To ban pesticides completely, we must acquire more agricultural
pesticides a case study was performed in Sri Lanka, where a large
lands to fulfill the food demands, which in turn will largely reduce
sample size was considered. Around 13,000 patients were admitted to
forest’s canopy and eventually will destruct the flora and fauna. The
hospitals due to pesticide poisoning in a single year. Toxicity has oc-
best notion to decrease the damages caused by these chemicals is to
curred due to both intentional and unintentional exposures. Each year
investigate narrow-spectrum pesticides and natural agents such as
about a thousand people lose their life due to pesticide poisoning in Sri
bacteria, viruses which have ultimate specificity. The alternative ap-
Lanka. A majority of patients (73%) intentionally consumed these
proach is to use insect hormones such as juvenile hormone and molting
chemicals, while the remaining (24.9%) were accidental or occupa-
hormone and their derivatives as insecticides, which could be an al-
tional exposures. Most common pesticides involved in such poisonings
ternative strategy to avoid resistance in insects and minimize effects on
were organophosphorous (Jeyaratnam et al., 1982).
non-targeted species including humans. In the present article, we have
reviewed the abnormalities associated with pesticides with special
emphasis on cancer, PD, AD and pesticide-mediated disorders of re-
2.2. Occupational exposure
spiratory and reproductive tracts and epigenetic modifications.
Occupational exposures include people engaged in pesticide in-
2. Exposures to pesticides
dustries, in transportation of pesticides, dealers, farmers, applicators
and sellers of fruits and vegetables in the markets. One of the pesticides
Pesticides cause risk to our health because majority of human po-
occupational outbreak has occurred in Pakistan, where 2800 workers
pulation gets exposed to pesticides actively or passively. The way we
involved in spraying Malathion to control malaria vectors were in-
get exposed to pesticides is also a matter of concern as it also decides
toxicated. During studies, it was found that these patients had de-
the dose of exposure. The dose is an important factor, as a Roman
creased cholinesterase activity in red blood cells due to organopho-
physician also considered “"the father" of toxicology credited with the
sphate poisoning (Baker et al., 1978). Paternal employment in
classic toxicology proverb, "All things are poison and nothing is without
agriculture and their exposure to pesticides has been associated with
poison; only the dose makes a thing not a poison". Exposure to pesti-
the development of cancers in their children (Fear et al., 1998).
cides could be broadly classified into three main categories, namely

104
A. Sabarwal et al.
2.3. Non-occupational exposure
All other exposed populations are mainly at the consumer level
(through fruits, vegetables, grains, etc.) and are considered as non-oc-
cupational exposures. The latter comprises the majority of the human
population and animals exposed to pesticides. It is difficult to directly
relate the exposure to pesticides and their hazardous effects. The eva-
luation becomes tighter when it comes to studying low-level long-term
exposures. Exposure to pesticides can also be classified on the basis of
acute and chronic toxicity (1972).
3. Cancers associated with pesticides
Studies have revealed the close association of pesticides and the
development of cancers in both children and adults. People who are
closely associated with pesticides exposure were found to be at greater
risk to various malignancies such as leukemia, Burkitt lymphoma,
neuroblastoma, Wilm’s tumor Non-Hodgkin lymphoma, soft tissue
sarcoma, ovarian cancer, cancers of lung, stomach, colon, bladder and
rectum (Bonner et al., 2017; Dich et al., 1997; Polanco Rodriguez et al.,
2017; Schinasi and Leon, 2014; Xu et al., 2010).
3.1. Childhood cancers
Many childhood cancers are found to be associated with pesticide
exposure. Compared to other cancer types, more convincing evidence
was presented in a population-based case-control study of acute mye-
locytic leukemia (AML). A comparison of 491 cases between an age
group of 0–9 years was done for polymorphisms in CYP1A1, CYP2D6,
GSTT1, and GSTM1 genes responsible to encode enzymes responsible to
metabolize carcinogenic substances (Infante-Rivard et al., 1999). En-
zymes of Cytochrome P-450 family are involved in the transformation
of pro-carcinogenic compounds to reactive species which have geno-
toxic and cytotoxic effects. Glutathione-S-transferases is one of the
enzymes which gradually inactivates these reactive species and many
pesticides for e.g. lindane, parathion, chlorophenol, atrazine use these
enzymes as substrates (Guengerich and Shimada, 1998). It is found that
mothers who got exposed to pesticides during pregnancies and if her
child carries a CYP1A1m1 or a CYP1A1m2 mutation had increased risk
of AML (Infante-Rivard et al., 1999).
A similar study also found a significant association between ma-
ternal exposure to pesticides and childhood leukemia. These women
were working in agriculture fields during pregnancy and were exposed
to pesticides (Kumar et al., 2014). The AHS (Agriculture Health Study)
also revealed childhood cancer which was significantly linked to the
parents who were engaged in pesticide applications in the fields. The
risk factor was increased [standardized incidence ratio = 1.36] (Roy
et al.). Risk of all lymphomas combined was also increased (SIR =
2.18), as was the risk of Hodgkin’s lymphoma (SIR = 2.56) (Flower
et al., 2004).
3.2. Prostate cancer
Environmental endocrine disrupting chemicals (EDCs) such as var-
ious industrial chemicals and pesticides are released into our environ-
ment and pose serious health problems. Increase incidence of hormone-
dependent cancers such as breast, testis, prostate and of the male re-
productive system have been associated with the hormone disrupters
(Skakkebaek, 2002). The prostate cancer (PCa) accounts for ∼25% of
cancer incidence and is the second leading cause of cancer-related
deaths in the male population in the United States (Roy et al., 2009).
2, 4-dichlorophenol (DCP) a metabolite of 2, 4-di-
chlorophenoxyacetic acid (2,4-D) are extensively used in agriculture
settings to control weeds. These herbicides have shown endocrine dis-
ruption activity. When cells were treated with these compounds alone
and in combination with 5α-dihydrotestosterone (DHT) at
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Environmental Toxicology and Pharmacology 63 (2018) 103–114
concentrations as low as 10 nM have been shown to stimulate cell
proliferation by 1.6 fold in prostate cancer cell lines. No such effect was
observed when treated with DHT alone. These chemicals also increased
the localization of androgen receptor in the nucleus from the cytosol
(Kim et al., 2005), suggesting DCP may act as a tumor promoter. A
significant association between prostate cancer risk and use of simazine
(OR = 1.89; 95% CI:1.08-3.33 for high exposure), lindane (OR = 2.02);
95% CI: 1.15–3.55 for high exposure and DDT (OR = 1.68 ; 95% CI:
1.04–2.70 for high exposure) was found, additionally the study also
reported significant excess risk for several constituents such as ma-
lathion, endosulfan, dichlone, carbaryl, 2,4-D and 2,4-DB (Band et al.,
2011).
In a prospective cohort study in Iowa and North Carolina, 45 rou-
tinely used pesticides were studied; the investigation included very
large groups (55,332) of pesticide applicators. A prostate cancer stan-
dardized incidence ratio of 1.14 (95% confidence interval: 1.05, 1.24)
was observed in the Agricultural Health Study cohort. Interestingly, it
was found that chlorinated pesticides and methyl bromide were asso-
ciated with increased risk of prostate cancer, but for other pesticides,
greater risk was observed for people with a family history of prostate
cancer. No significance was found in people without a family history
(Alavanja et al., 2003).
Chlorpyrifos (CPF) is an organophosphate pesticide and is widely
used in agricultural fields. Organophosphates are metabolically acti-
vated and are irreversible inhibitor of cholinesterases and act as neu-
rotoxins (Amitai et al., 1998). Chlorpyrifos when given to mouse with
PTEN deletion makes the animal predisposed to prostate cancer.
Chronic exposure for a period of 32 weeks to chlorpyrifos did not
promote prostate cancer in animals but reached sufficient levels to in-
hibit acetylcholinesterase activity in plasma (Svensson et al., 2013),
suggesting more investigations are needed to conclude CPF as a carci-
nogen in prostate cancer. In a meta-analysis, increased risk of prostate
cancer was studied in the farmers associated with a contaminated
pesticide with highly toxic 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin
(TCDD). Five studies till 2006 were taken to investigate 26,706 people
exposed to the pesticide which showed a positive correlation with
pesticide exposure and death due to prostate cancer (Kabir et al., 2018).
Urokinase (uPA) and its receptor (uPAR) are important regulators of
cell proliferation, invasion and migration. Effect of herbicide, roundup
and insecticides lorsban and warrior were studied on the expression of
uPA and uPAR in transformed prostate epithelial lines. It was found that
roundup, lorsban and warrior induced the expression of uPA while
lorsban and warrior caused modulation of uPAR. Interestingly, when a
combination of all were used the induction of uPA and uPAR were
stronger compared to alone treatments (Potti and Sehgal, 2005).
3.3. Breast cancer
Exposure to persistent organic pollutant’s which includes o,p'-di-
chlorodiphenyltrichloroethane (o,p'-DDT), and its metabolites p,p'-di-
chlorodiphenyldichloroethylene (p,p'-DDE), and p,p'-di-
chlorodiphenyldichloroethane (p,p'-DDD) and other related pesticides
are common due to the increased load of chemical pollutants in the
environment. POPs are known endocrine disruptors in humans. To in-
vestigate their role in breast cancer cells, a study was done with two
breast cancer cell lines MCF-7 (positive for estrogen receptor-α) and
MDA-MB-231 (negative for estrogen receptor-α). With the concentra-
tions, 50 nM to 1 μM differential effect was seen with decrease in cell
proliferation and viability in MCF-7 cells without any change in MDA-
MB-231 cells. Further, cell invasion was induced in MCF-7 cells whereas
it was reduced in the advanced and more invasive cell line MDA-MB-
231 cells (Pestana et al., 2015).
Another herbicide atrazine has been evaluated for its carcinogenic
effect on breast cancer. Oncogenic studies on Sprague-Dawley (SD) rats
revealed decreased latency and an increase in the prevalence of ade-
nocarcinoma and fibroadenoma of mammary tumors, these findings
A. Sabarwal et al.
were not consistent in Fischer-344 rats. Interestingly, atrazine-induced
mammary tumors occurred only in aging female SD rats. This was
particularly due to suppression of luteinizing hormone rush thus, a
stage of persistence in estrus cycle has occurred. Gradually estrogen and
prolactin remain at high levels in the blood. Such actions will not be
liable for the human system due to low levels of estrogen and prolactin
after reproductive senescence. Epidemiological studies do not support
any cancer-causing role between atrazine and breast cancer. Studies
show consistency with the USEPA classification as atrazine “not likely
to be carcinogenic”(Simpkins et al., 2011). In another study, a modest
increase in the risk of breast cancer was found to be associated with
acute events in a subgroup of young females who were exposed during
childhood and adolescence (Niehoff et al., 2016). A risk study was
conducted in the spouses of pesticide applicators in an Agriculture
Health Study. Among 30,003 women, 25.9% reported the use of orga-
nophosphate (OP) pesticides and 718 women exposed to OPs were di-
agnosed with cancer during the follow-up period. Organophosphate use
was associated with an elevated risk of breast cancer (RR = 1.20, 95%
CI 1.01–1.43), one of the most commonly used OP, malathion was as-
sociated with the elevated risk of thyroid cancer and use of diazinon
was positively associated with increased risk of ovarian cancer
(RR = 1.87, 95% CI 1.02–3.43) (Lerro et al., 2015)
3.4. Colorectal cancer
Colorectal cancer (CRC) is the second leading cause of cancer-re-
lated deaths in the United States men and women combined (Raina
et al., 2016). In an Agriculture Health Study (AHS), an investigation
was done to elucidate the relationship between pesticides and color-
ectal cancer. Most of the pesticides were not found to have an asso-
ciation with colorectal cancer. For rectal cancer, chlorpyrifos has shown
a significant exposure-response pattern that was increased by 2.7 fold.
Aldicarb was found to be significantly associated with colon cancer and
highest exposure increased the risk by 4.1 fold (95% confidence in-
terval: 1.3–12.8). However, strong evidences are lacking to prove a
close association between these pesticides and colorectal cancers which
requires further studies with detailed mechanisms (Lee et al., 2007).
Another similar study revealed an unconvincing association be-
tween extensively used herbicide dicamba and colon cancer (Samanic
et al., 2006). A study was done to investigate the long-term health ef-
fects, including carcinogenic effect, of organochlorine pesticides diel-
drin and aldrin. A total of 570 employees, who were involved in the
production plants of these pesticides from January 1954 to January
1970 were followed for specific mortality until January 2001. An
average intake of 737 mg for dieldrin was recorded in blood levels of
343 workers. And, 171 workers died before 2001 compared with an
expected number of 226.6, giving a standardized mortality ratio (SMR)
of 75.6. This deficit in total mortality was mainly due to a deficit in
cardiovascular disease mortality, but not due to cancer mortality. The
number of rectal cancers was significantly higher, however, this rela-
tion was observed in low-intake subgroups and thus it appears that
there is no relationship between exposure to these pesticides and cancer
incidence (Swaen et al., 2002). To estimate the risk of cancer, a po-
pulation-based case-control study was conducted among people re-
siding in 10 districts of Andalusia (Spain). The districts were categor-
ized as higher or lower uses of pesticides based on the number of
hectares of land used for intensive agriculture and sale of pesticides per
capita. It was found that the prevalence rates per 100,000 population
for cancers of stomach, colorectal, liver, bladder and brain were sig-
nificantly greater in the regions with higher use of pesticides as com-
pared to regions of lesser use of pesticides (Parron et al., 2014). Effects
of maneb, mancozeb and zineb were studied on transformed and non-
transformed colon cells. Mancozeb and maneb were toxic to HT-29,
CCD-18Co and Caco2 cells. However, zineb did not caused any sig-
nificant decrease in cell viability. The toxicity was due to the metal
moiety present in mancozeb and maneb. (Hoffman and Hardej, 2012).
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Environmental Toxicology and Pharmacology 63 (2018) 103–114
3.5. Non-Hodgkin lymphoma (NHL)
Non-Hodgkin lymphoma is a diverse group of malignancies which
affects lymph and immune system; it consists of more than 20 different
malignancies. In the past few decades, this particular type of malig-
nancy has been increased worldwide (Alavanja and Bonner, 2012).
Phenoxyacetic acid herbicides, in particular, 2,4-dichlorophenoxyacetic
acid is used frequently in agricultural settings and are found to be as-
sociated with 2-8-fold increase of NHL in studies conducted in Sweden,
Kansas and Nebraska (Zahm and Blair, 1992; Zahm et al., 1990).
Growing evidence indicate that exposure to organochlorine pesticides
(OCPs) increases the risk of developing NHL. In a meta-analysis, the risk
to pesticide exposure for NHL was studied, positive associations for
dichlorodiphenyldichloroethylene, hexachlorocyclohexane, chlordane,
and hexachlorobenzene were reported (Luo et al., 2016). Similarly, a
positive association was found in another meta-analysis performed with
organophosphate pesticides (OPs). In this study three non-halogenated
OPs Terbufos, Malathion and Diazinon were selected within a period of
1985–2017. Diazinon showed a significant positive relation with ex-
posure and NHL. It is reported that immune dysfunction is directly
related to NHL. Malathion attacked immune cells directly while dia-
zinon caused disruption of a neuro-immune system that involves a
cholinergic system of lymphocytes (Hu et al., 2017).
4. Other disorders associated with pesticides
It is generally believed that pesticides cause only life-threatening
diseases like cancer; however, interestingly there are many other dis-
orders which are associated with these chemicals. Some of these dis-
orders could be fatal if untreated and may compromise the quality of
life of individuals. Some important disorders are discussed here in brief.
4.1. Parkinson’s disease
Etiology of Parkinson’s disease (PD) is largely unknown. PD is an
idiopathic disease and its pathological feature is loss of pigmented
neurons in substantia nigra of the brain. Epidemiological studies have
shown environmental factors, including pesticides could play important
roles in the initiation of PD. Rotenone a broad spectrum herbicide is a
known inhibitor of mitochondrial complex I. It was found that chronic,
systemic inhibition of this complex by rotenone causes selective ni-
grostriatal dopaminergic degeneration, which is associated with partial
or complete loss of muscle movement due to disruption in the basal
ganglia. These findings indicated a resemblance with the features of PD
(Betarbet et al., 2000). In a case-control study, 36 commonly used
pesticides were studied. The study included 357 incident PD cases and
752 population controls living in the central valley of California. Re-
sults showed a strong relation between these pesticides and etiology of
idiopathic PD (Betarbet et al., 2000). In a similar case control study,
contribution of Nitric oxide synthase (NOS) gene and organophosphate
(OP) to PD risk was investigated. NOS enzyme produces nitric oxide
(NO), a pro-oxidant which can damage neurons, is considered as a
candidate for PD. For this study, 357 PD incident cases and 495 po-
pulation controls were taken into consideration. With the help of global
information system (GIS), the household and ambient agricultural OP
exposure were accessed. Eight NOS SNPs from the blood and saliva
samples of the above population were investigated. Their findings in-
dicated that the OP pesticides were more strongly associated with PD
among the participants having variant genotype of NOS1. This study
supports a role for NOS2A genetic variants in PD susceptibility and
NOS1 as a modifier of associations with PD in OP pesticide exposed
populations (Paul et al., 2016). Another fungicide, Benomyl, through its
metabolite thiocarbamate sulfoxide, inhibits aldehyde dehydrogenase
(ALDH), which in turn increases the accumulation of the reactive do-
pamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) causing
degeneration of dopaminergic neurons and thus, enhances the
A. Sabarwal et al.
pathogenesis of PD (Fitzmaurice et al., 2013). Most of the studies which
are done to find a correlation between pesticide exposure and PD have
significantly shown a positive relation. A study was conducted in
Netherlands to assess the risk of exposure to pesticides and PD. Higher
risk of PD was involved with rotating crops and paraquat was found to
be positively associated with increased risk of PD (Brouwer et al.,
2017). A meta-analysis found that pesticide exposure can cause genetic
alteration in the genes involved in the pathogenesis of PD. Exposure to
pesticides increased the risk of alterations in genes such as GST, PON-1,
MDR-1 and SNCA (Ahmed et al., 2017). A significant increase in 13 out
of 23 case-control studies that considered overall exposure to pesticides
was reported recently. An increased PD risk has been associated with
insecticides chlorpyrifos & organochlorines, herbicides like paraquat
and fungicide like maneb also increased the risk alone or in combina-
tions (Freire and Koifman, 2012). Various experimental data on rodents
also suggested the risk of PD when exposed to pesticides. Paraquat and
rotenone which are known to induce neuroinflammation and dopami-
nergic cell loss are the most common pesticides used to establish the
toxin-based animal model of PD. Swiss albino mice were exposed to
rotenone and frontal cortex, hippocampus and substantia nigra were
studied as these are the major regions involved in the pathogenesis of
PD. It was found that rotenone treatment caused a significant genera-
tion of ROS–scavenging enzymes, degeneration of DOPA decarbox-
ylase-positive neurons and fibers in dopaminergic as well as norepine-
phrinergic system, number of astrocytes decreased in these areas,
indicating rotenone is positively co-related with PD (Mitra et al., 2015;
Taetzsch and Block, 2013).
Systemic injection of paraquat to C57BL/6 mice acutely or once a
week for 4, 8, 12 and 24 weeks caused as dose-and age-dependent
neurodegeneration of dopaminergic neurons in the SNpc population,
whereas cells which produce GABA in substantia nigra and cholinergic
neurons were not affected (Baltazar et al., 2014). Similarly, a single
dose of paraquat did not affect the number of dopaminergic neurons,
however, repeated treatments of paraquat for 2 days significantly killed
dopaminergic neurons. These results suggested that chronic exposure to
paraquat would lead to the vulnerability of dopaminergic neurons
(Shimizu et al., 2003).
4.2. Alzheimer’s disease
Alzheimer’s disease (AD) is one of the most common causes of de-
mentia in aged people. The characteristic features of the disease include
the presence of extracellular amyloid–beta (Aβ) plaques, neuronal
death and the loss of synapses. Environment contaminants are found to
be positively associated with the pathogenesis of AD. Many studies have
found that chronically exposed individuals to pesticides have a high
prevalence of cognitive, behavioral and psychomotor dysfunction and
Alzheimer’s disease dementia. Organophosphate pesticides are found to
inhibit acetylcholinesterase similarly as the drugs used to treat AD,
have also shown to cause abnormalities in microtubule arrangements
and tau hyper-phosphorylation (Zaganas et al., 2013). Mild cognitive
impairment (MCI) is one of the prodromal stages of dementia and is
associated with the exposure to pesticides, every year 5–15% in-
dividuals diagnosed with MCI, develops AD (Petersen, 2011). In a case-
control study conducted by Parron et al. in Andalusia (Spain) which
involved 17, 429 subjects and data was collected between 1998–2005
using hospital records. Subjects were categorized in two areas, low
agricultural practice/use of pesticides and high agricultural practice/
high use of pesticides per capita. They have shown that subjects lived in
the areas with a higher amount of pesticides were at greater risk to PD,
AD and multiple sclerosis (Parron et al., 2011). In the cache county
study, among 3084 enrollees who did not have dementia previously,
some 500 individuals were found to developed incident dementia and
344 developed AD after exposure to pesticides. The Cox proportional
hazard model showed an increased risk to pesticide exposure was as-
sociated with all types of dementia with increased risk hazard ratio and
107
Environmental Toxicology and Pharmacology 63 (2018) 103–114
AD. The risk of developing AD is higher with organophosphate pesti-
cides than the risk associated with organophosphate pesticides (Hayden
et al., 2010).
4.3. Reproductive disorders
Exposure to certain pesticides in sufficient doses may increase the
risk of sperm abnormalities, decreased fertility aberrant abortions, de-
fects in birth and fetal growth retardation (Frazier, 2007). Carbosulfan,
a carbamate pesticide has shown an increase in chromosomal aberra-
tions (CA), bone marrow micronucleus formation (MN), and sperm
abnormality in mice. At all three acute doses used in the study (1.25,
2.5 and 5 mg/kg) there was a concentration-dependent increase in the
CA, micronucleated polychromatic erythrocytes (PCEs) and sperm head
abnormalities, but did not affect the total sperm count. These findings
indicate carbosulfan as a potent genotoxic agent and could also act as a
potent germ cell mutagen (Giri et al., 2002). Cypermethrin, a pyre-
throid insecticide was evaluated for its toxic potentials on reproductive
and fertility parameters in the adult male Sprague-Dawley rats. These
animals were exposed to tap water containing different doses of cy-
permethrin in ppm concentrations for 12 weeks. A significant decrease
in fertility was recorded in male rats, which ingested cypermethrin at a
concentration of 13.15 and 18.93 mg/per day. A significant reduction
in the number of viable fetuses has occurred in females impregnated by
the treated male rats. A weight gain in testis and seminal vesicles and
decrease in epididymal and testicular sperm counts was also observed
in the treated animals (Elbetieha et al., 2001; Rios et al., 1995). In
another similar study, 15 day intact adult assay was performed to in-
vestigate the reproductive toxicity of cypermethrin, male mice were
given different concentrations of cypermethrin through oral gavage for
15 days. Rats were euthanized and their epididymis, testis, seminal
vesicles, and prostate glands were excised. The study found that the
weighed of prostate and sperm production were significantly decreased
in cypermethrin treated mice. Also, the morphological changes in-
cluding necrosis was recorded in seminiferous tubule, sertoli cells. To
study the underlying molecular mechanism, expression of androgen
receptor and levels of testosterone were analyzed and both were sig-
nificantly reduced in rats fed with cypermethrin. Thus cypermethrin
has potential to induce impairments of the structure of seminiferous
tubules and spermatogenesis in male rats (Hu et al., 2013).
Another extensively used, non-selective herbicide paraquat, has also
shown to cause genotoxicity in mouse somatic and germ cells. The
mouse were exposed singly or multiple times. Single time exposure did
not cause any chromosomal aberration (CA), but with multiple ex-
posures there was an increase in CA. The sperm shape abnormality was
also increased when animals were treated at three stages of cell de-
velopment:spermatozoa, spermatid and preleptotene spermatogonial
cells (Rios et al., 1995). Incidence of miscarriages or fetal losses were
significantly increased in spouses of fungicide applicators. Surprisingly,
significant deficit in the number of male children born to the spouses of
fungicide applicators was also observed (Garry et al., 2002). A popu-
lation based-case-control study was carried out on pregnant women and
male children living in high risk and low risk areas in Andalusia
(Spain). It was found that prevalence rates and risk of miscarriages, low
birth weight, hypospadias, cryptorchidism and micropenis were sig-
nificantly higher in the areas where pesticides were used in higher
amounts (Garcia et al., 2017).
4.4. Respiratory disorders
In the 1700s, Bernardino Ramazzini was one of the first researchers
who informed the risk of respiratory disorders are greater in farmers
(Hoppin et al., 2006). In developing countries like India, most of the
pesticide applicators do not use Personal Protective Equipments (PPE) like
safety masks, gloves, etc. which brings them into direct contact and higher
doses of pesticide exposure. As most of the pesticides are sprayed in the
A. Sabarwal et al. Environmental Toxicology and Pharmacology 63 (2018) 103–114

Fig. 2. Pesticide modulates various cellular and sub-cellular activities leading to genetic and epigenetic changes and cause various diseases or cell death. Pesticide
may cause endocrine disruption and act agonistically by binding to hormone receptors present on cell membrane or nucleus, causing disruption of cellular signaling.
Pesticides can also induce inflammatory signals or can generate reactive oxygen species (ROS) which can cause oxidative stress leading to accumulation of unfolded
protein aggregates through deforming ubiquitin proteasome system and may cause genetic and epigenetic modifications leading to various diseases. Toxicity of
pesticide may disrupt the cellular functions by causing malfunctioning of mitochondria and endoplasmic reticulum.

fields, it generates aerosols that can directly enter into the respiratory
system of these agricultural workers. A cross-sectional study conducted in
North India has shown convincing results that pesticides causes diverse
respiratory illnesses which includes wheezing, dry cough, irritation of the
respiratory tract, blood in sputum etc (Fareed et al., 2013).
In another study, which included 98 farmers and 98 nonfarmers, a
comparison of dermo-respiratory symptoms from lung-function spi-
rometer values and serum IgE levels were done. The spirometer tests
measured in the farmers were significantly reduced while they had high
levels of serum IgE. The study also revealed significant differences in
dermo-respiratory symptoms such as cough, pharyngitis, bronchitis,
asthma, respiratory insufficiency, pneumonia, dyspnea, nasal catarrh,
sinusitis, pharyngeal irritation, nasal irritation (dryness, sneezing and
secretions), ocular irritation, cutaneous pruritus, and contact dermatitis
(Bener et al., 1999). Organophosphate and carbamate exposure is found
to decrease red blood cell cholinesterase in 77% of pesticide applicators
while 27% had a decrease in plasma cholinesterase levels (Rama and
Jaga, 1992). To evaluate the association between prenatal exposures to
organochlorine pesticides and childhood asthma, four studies were
conducted in Spain. 482 mothers during antenatal care and 405 chil-
dren after birth till the age of 6 were followed. The researchers found
that higher concentration of DDE (median = 1.03 ng/mL), a metabolite
of DDT, in the cord serum at birth was positively associated with in-
creased risk of wheezing at 4 years of age (relative risk 2.36, 95% CI
1.19–4.69) for the highest quartile (> 1.90 ng mL−1). In addition to
DDE, hexachlorobenzene (0.68 ng/mL) and polychlorobiphenyls
(0.69 ng/mL) was also detected, which were significantly associated
with clinical asthma (Mamane et al., 2015; Sunyer et al., 2006, 2005). A
cross-sectional study conducted with the workers in a bottling plant
taken as control and compared with the workers involved in pesticide
processing plant. It was found the latter were at significantly much
higher risk of developing respiratory disorders including chronic cough,
dyspnea grades 3 and 4, throat irritation, nasal catarrh, and nasal
dryness (Zuskin et al., 2008)
5. Molecular mechanism linked to pesticide-induced chronic
disease
The toxic effects that pesticides on human health have been widely
studied in the last few decades. Observational studies on workers ex-
posed to pesticide along with animal models have shown that how these
chemicals are responsible for detrimental effects on health. However,

108
A. Sabarwal et al.
the understanding of the molecular mechanism by which pesticides
could impact on human health is of great importance. The environ-
mental factors such as pesticides that may regulate and stimulate the
disease processes are important thus, understanding of these funda-
mental changes will drive the scientific basis for health decisions. The
multiple mechanisms that cells adapt to respond and to cope up with
the environmental signals include communication pathway or signal
transduction pathway. Various sensors or receptors sense the presence
of foreign compounds surrounding the cells and induce a cascade of
events that are intended to lead to neutralization and excretion of these
compounds. In many cases, the metabolism of these compounds give
more toxic metabolites or induce Reactive Oxygen Species (ROS) that is
even more harmful to cells. Moreover, the metabolism of these com-
pounds disturbs the other essential processes which further disturb the
overall health. The mechanisms of action of pesticides have been stu-
died at the molecular level. There are various pathways which are being
followed by pesticides to pose their detrimental effects at cellular level.
Some of the most studied mechanisms are being discussed here:
5.1. Endocrine disruption
Endocrine disruption is one of the important targets of these pesti-
cides. Endocrine disrupting chemical (EDC) is best defined as “an
exogenous substance that causes adverse health effects in an intact
organism, or its progeny, secondary to changes in endocrine function”
(European workshop on the impact of endocrine disrupters on human
health and wildlife [EEC], 1996). Endocrine hormones are chemical
substances secreted by a particular gland and they act on distantly lo-
cated cells, tissue, organ, and organ system, these hormones are very
specific in nature, additionally, their concentration and the time of
release on specific growth period is of great importance. Pesticides
disrupts their activity, time of release or can mimic these hormones
(Fig. 2) which may result in reduced fertility, male and female genital
tract abnormality (Baskin et al., 2001; Kabir et al., 2015), abnormalities
of thyroid glands (Akhtar et al., 1996), compromised immune function
and various types of cancer (Garry, 2004; Mathur et al., 2002).
Most of the reported harmful effects of EDCs are attributed to their
interference with hormonal signaling mediated by nuclear hormone
receptors (NRs) (Swedenborg et al., 2009; Toppari, 2008). Most im-
portantly three transcription superfamily are responsible for the reg-
ulation of xenobiotic metabolizing enzymes namely basic-helix-loop-
helix/Per-ARNT-Sim proteins, nuclear receptors and basic leucine
zipper proteins (Kliewer and Willson, 2002). Pesticides can bind and
activate various other hormone receptors such as androgen receptors
(AR), estrogen receptors (ER) (Tabb and Blumberg, 2006), constitutive
androgen receptor (CAR) (Tolson and Wang, 2010), estrogen-related
receptor (Musicco et al.), Aryl hydrocarbon receptors (AhR), pregnane x
receptors (PXR), and act agonistically to the natural hormone action. In
other cases, pesticides may bind the receptors without activating them
which blocks the receptors and inhibits their normal action. The en-
docrine disrupting pesticides such as mancozeb, maneb, zineb, ziram,
thiazopyr, pyrimethanil, amitrole, cyhalothrin, prodiamine and fipronil
are found to interfere with synthesis, metabolism, transportation, and
elimination of hormone which results decrease in natural hormone
concentration (Akhtar et al., 1996; Cocco, 2002; Leghait et al., 2009;
Sugiyama et al., 2005). In a ‘two-generation rodent study’, in which two
generation of rodents were studied for reproductive disorder induced
by pesticides such as alterations in all the aspects of male and female
reproductive performances, from ovarian or testicular abnormalities to
disrupted endocrine functions. These studies provide relevant data to
understand a gender-specificity because the same toxicant can produce
very different results depending on the endocrine pathways affected.
Certainly, a chemical that impairs pituitary gonadotropin secretion may
interfere with estradiol or testosterone production in a very different
way in males and females (Cecconi et al., 2007). Generally, effects are
studied for single pesticides in lab animals, however, an interesting
109
Environmental Toxicology and Pharmacology 63 (2018) 103–114
study has employed a mixture of pesticides as humans get exposed to
combination of different pesticides. Combination of pesticides even at
very low levels, no observed adverse effect levels (NOAEL), were given
to male rats and found that pesticides can cause adverse effects on male
sexual development even below NOAEL. In the experiments, oral ga-
vage was given to rats for 5 times with a mixture of procymidone,
mancozeb, epoxyconazole, tebuconazole and prochloraz, at con-
centrations below NOAEL. The mixture of pesticides caused impaired
parturition leading to significant decrease in the number of live-born
offspring, higher anogenital distance in both male and female pups,
malformations of genital tubercle in male pups, higher nipple retention,
decreased epididymis and prostate at 13th day. The study also showed
that when these pesticides were used alone, no such effects were ob-
served (Jacobsen et al., 2010).In utero exposures to pesticides is the first
point that can affect the maternal-placental-fetal balance. A prospective
study was conducted on pregnant women living in rural areas of Rio
Negro province where organophosphate pesticides are applied ex-
tensively throughout six months per year. Blood samples were obtained
and analyzed for organophosphate biomarkers including cortisol, cho-
linesterases, β-glucuronidase, progesterone, and glycemia. Aspartate
aminotransferase (AST), alanine aminotransferase (ALT), and liver
function was assayed by albumin for liver injuries. Subjects were fol-
lowed during pre-spray and spraying periods of pesticides. It was found
that the plasma and erythrocyte cholinesterase decreased significantly
(p < 0.01) during the spraying period. Additionally, levels of cortisol
increased significantly in the first trimester of pregnancy in spraying
compared to pre-spraying periods. Also, levels of alanine amino-
transferase and the ratio of AST/ALT increased markedly, indicating
sub-clinical hepatotoxicity (Cecchi et al., 2012). The present data in-
dicate that pesticides are potent endocrine disruptors’ mainly in mixed
forms and NOAEL concentration should be studied again and new
guidelines should be made. Herbicide pretilachlor has shown to cause
endocrine disruption at early stage of development of zebrafish. It has
caused upregulation of p53, Mdm2 and Bbc3 and increased activities of
caspases involving apoptosis in zebrafish (Jiang et al., 2016). Similarly,
effect of azocylotin was studied on the metamorphosis of Xenopus laevis,
it was found that azocyclotin downregulated the expression of genes
involved in thyroid hormone signaling which decreased the level of
thyroid hormones and thus interfered with the metamorphosis of Xe-
nopus (Li et al., 2016).
5.2. Genetic damages
Genetic damages caused by pesticides can be broadly classified into
three major classes namely i) Pre-mutagenic damages like DNA strand
breaks (Grover et al., 2003; Lebailly et al., 1998), DNA adducts (Peluso
et al., 1996) ii) gene mutations like insertions, deletions, inversions and
translocation (Edwards and Myers, 2007) iii) chromosomal aberrations,
including loss or gain of whole chromosome (aneuploidy), deletion or
breaks (clastogenicity), and chromosomal segments or rearrangements
(Carbonell et al., 1993).
It is widely established fact that ROS production is associated with
DNA damage, lipid peroxidation (Banerjee et al., 1999) and change in
mitochondrial membrane potential (Bonsi et al., 2004). Enzymes in-
volved in pesticide detoxification produces electrophilic compounds
which are carcinogenic epoxides, these events may also render the re-
pair machinery less efficient resulting in premature aging and apop-
tosis. ROS imbalances may also recruit aberrant proteins which may
results in imbalance of the signaling pathway leading to tumorigenic
processes (Wang, 2008). Exposition to different chemical insults present
in our environment may be a cause of mutagenic origin of cancer
(Claxton and Woodall, 2007). Genes and/or gene products involved in
metabolism of xenobiotic compounds can be modulated by such mu-
tations. These changes at genetic level may produce polymorphisms
resulting in altered affinity to their ligand or may change the expression
of downstream target genes (Dong et al., 2008).
A. Sabarwal et al.
5.3. Oxidative stress
Living in an oxygenated environment is very challenging, it took
millions of years for the living organisms to evolve effective strategies
to detect and neutralize metabolites of molecular oxygen, which are
known as ROS (Finkel and Holbrook, 2000). Oxidative stress is a con-
dition when there is an imbalance between pro-oxidant and antioxidant
homeostasis that results in the formation of toxic reactive oxygen spe-
cies (Barnham et al., 2004). These ROS can cause damages to lipids,
proteins and DNA (Sabarwal et al., 2017). ROS are very unstable with
very high reactivity due to unpaired electrons, to gain electrons; they
attack nearby molecules which results in their damage. All the nearby
biomolecules are susceptible for their attack, but lipids get severely
affected (Li and Zhang, 2013; Morgan et al., 2007). Generation of ROS
is associated with the risk of cancer. ROS can influence the expression
of many genes involved in inflammation; cell transformation; and
tumor cell death or survival, proliferation, invasion, angiogenesis, and
metastasis. ROS is found to activate the transcription factors NF-κB,
activator protein-1 (AP-1), HIF-1α, signal transducer and activator of
transcription 3 (STAT3) (Gupta et al., 2012). Polymorphisms in genes
related to oxidative stress (e.g. mangano-type superoxide dismutase,
catalase, or glutathione peroxidase) may not be directly related to risk
to cancer instead the defective protection machinery against oxidative
stress may results to the disease progression (Ryter et al., 2007). Pi-
peronylbutoxide (PBO) is an extensively used pesticide (Muguruma
et al., 2007) and efficiently up-regulates CYPA1, one of the enzymes
involved in catabolizing pro-carcinogens (Canistro et al., 2002). In mice
PBO has a liver-tumor-promoting effect through increased ROS pro-
duction which is a byproduct of hepatic microsomal oxidation
(Muguruma et al., 2007). Oxidative stress is also found to be closely
associated with the pathogenesis of many neurodegenerative diseases
like Parkinson’s disease (PD) and Alzheimer’s disease (AD). ROS is also
found to be associated with ageing, chronic inflammation and cancer
(Reuter et al., 2010). One of the modes for the toxicity of organopho-
sphate pesticides is the formation of ROS. Cytochrome P450 metabo-
lizes these chemical, P450 enzymes are basically monooxygenases
which catalyze the oxidation through the addition of one atom of
oxygen to their substrate by an electron transport pathway (Ahmad
et al., 2010).
Another mode of ROS generation is due to increase in energy de-
mand by the cellular machinery and inhibition of oxidative phosphor-
ylation, which in turn induces glycogenolysis in the liver, which is re-
quired to fulfill cell’s energy requirement, these all add up in the ROS
generation (Milatovic et al., 2006; Rahimi and Abdollahi, 2007). A
study was done on mouse lung fibroblast cells using phosphamidon and
dieldrin, to investigate their clastogenic properties through the gen-
eration of ROS. Phosphamidon induced DNA damage was reduced by
catalase, while glutathione peroxidase has a protective role against
damage induced by both phosphamidon and dieldrin. Involvement of
these antioxidant enzymes shows that oxidative stress and damage is
involved with these two pesticides (Cicchetti and Argentin, 2003). In
another study cypermethrin and fenvalerate was fed orally to rats for a
single time, results indicated that these chemicals increased lipid per-
oxidation in erythrocytes within 3 days of administration of these
pesticides. The increased levels of oxidative enzymes such as catalase,
superoxide dismutase and reduced glutathione in erythrocytes point out
the involvement of oxidative stress occurred due to these pyrethroid
pesticides (Kale et al., 1999). Workers of North Indian population are
exposed to commonly used insecticides. A study was done on 70
healthy, pesticide applicators that were exposed to pesticides for 5
years and compared with 70 controls. The investigation was focused on
the level of antioxidant enzymes (superoxide dismutase, catalase, glu-
tathione-S-transferase and glutathione peroxidase), acet-
ylcholinesterase (AChE), lipid peroxidation and glutathione (GSH) in
their red blood cells (RBC).The levels of GSH, RBC-AChE activity were
significantly decreased when compared with controls. Reduced AChE
110
Environmental Toxicology and Pharmacology 63 (2018) 103–114
levels in blood were alarming and neurotoxicity may be associated with
these workers. In addition to a decrease in the level of GSH in RBCs
accompanied with an increased level of other anti-oxidative enzymes
and there was a significant level of overexpression of CYP2E1 and GST-
pi was observed, which is a marker of oxidative stress (Sharma et al.,
2013).
Paraquat is second most widely used herbicide and is known to
accumulate in human lungs and causes oxidative stress-mediated fi-
brosis. Paraquat was found to cause toxicity in normal human bronchial
epithelial cells (BEAS-2B), leading to oxidative stress-mediated mi-
tochondrial damage and cell death in concentration-dependent fashion.
Paraquat also caused the production of inflammatory and profibrogenic
factors including tumor necrosis factor α, interleukin 6, and trans-
forming growth factor β1, that lead to the transformation of normal
human lung fibroblasts (WI38-VA13) to myofibroblast. These effects
were significantly inhibited by resveratrol, it caused activation of Nrf2
signaling pathway, which in turn induced the expression of antioxidant
response element-dependent cytoprotective genes. Furthermore, know-
down of Nrf2 significantly induced paraquat-induced cytotoxicity and
abolished protection by resveratrol. The findings of the studies de-
monstrated that resveratrol attenuated paraquat-induced ROS genera-
tion, inflammation and fibrotic reactions by the induction of Nrf2 sig-
naling (He et al., 2012; Jablonska-Trypuc et al., 2017). In a case study,
oxidative stress in placenta of females due to organophosphate ex-
posure during pesticide pulverization was studied. Placental samples
were collected from females residing in the agricultural areas during
the periods of pesticides pulverization, non-pulverization and control
groups. Significant decrease in carboxylesterase activity during pul-
verization was recorded, additionally an inverse association between
catalase activity and placental index was also found. Placental index is a
useful metric for estimating placental inefficiency (Chiapella et al.,
2014). Sub-lethal-doses of quinalphos for longer durations (15, 30 and
90 days) to adult male rats caused significant decrease in mal-
ondialdehyde, glutathione-s-transferase activity together, moreover, it
also caused decrease in ferric reducing ability of plasma and glu-
tathione. Quinalphos caused histoarcheitectural alterations in liver in-
cluding dilation of sinusoids, changes in focal fatty, accumulation of
eosinophils and single cell necrosis (Subramaneyaan et al., 2012).
5.4. Epigenetic effects
Epigenetics is the study of heritable changes in gene expression that
occurs without any change in the DNA sequence. Environmentally in-
duced changes in gene regulatory mechanisms co-relate with many
diseases including cancer (Tabrez et al., 2014). Epigenetics majorly
consist of changes in the DNA methylation patterns, histone modifica-
tions and differential expression of non-coding RNAs, which causes
variations in gene expression. Research in this area has revealed many
important relations between epigenetic changes and pathogenicity of
several diseases including cancer. Environmental factors are found to be
associated with epigenetics. Evidence from animal studies supports the
role of environmental epigenetics in disease susceptibility (Jirtle and
Skinner, 2007). Pesticides are environmental contaminants and now
these chemicals are found to be associated with epigenetic modifica-
tions thus pertaining effects on human health (Table 1).
Shutoh et al., 2009 experiments’ on young rat, in which DDT ex-
posure alters the methylation pattern in the hypothalamus, showed that
6 CpG islands in Sst, Gal, Ttr, Arf1, Msx1 and Grifin genes were sig-
nificantly hypomethylated compared with untreated groups. The mal-
functioning of DNA methylation machinery is augmented by low levels
of ROS, which leads to incomplete methylation of specific gene regions
(Shutoh et al., 2009). Recently, with the use of modern techniques, the
degree of DNA methylation at different site across the genome have
been measured and shown to be related with the degree of methylation
changes during aging and that differential methylation at certain site is
linked with age. Based on the retrieved information on the degree of
A. Sabarwal et al. Environmental Toxicology and Pharmacology 63 (2018) 103–114

methylation at such sites, DNA methylation age can be calculated, in-

(Wahlang et al., 2016)

(Rusiecki et al., 2008)
(Stouder and Paoloni-

(Shutoh et al., 2009)

(Anway et al., 2005)
(Mishra et al., 2009)
(Marsit et al., 2006)

(Wang et al., 2010)

(Wang et al., 2010)
creased calculated DNA methylation age compared with chronological

(Song et al., 2010)

(Song et al., 2011)

(Ross et al., 2010)

(Kuo et al., 2008)
Giacobino, 2011)
(Li et al., 2011)
age has been linked with poor survival rate (Marioni et al., 2016, 2015;
Perna et al., 2016). A few epidemiological studies conducted to ex-
amining the link between OCPs and aging; the exposure of oxychlor-
dane and TNC was found to affect telomere length and may contribute
Ref.

to an accelerated ageing (Guzzardi et al., 2016). Based on the above

findings, a cross sectional study was performed by Lind et al., 2018,
Porcine kidney epithelial cell line (PK15)

Human umblical vein endothelial cells

they established the relation with increased OCPs and increased DNA

Human Gastric adenocarcinoma cells

Human immortalized lymphoblast

methylation age (Lind et al., 2018).

Human colon epithelial-FHC cells

Immortalized rat mesencephalic/
dopaminergic cells (N-27 cells)

Methylation of Alu and long interspersed nucleotide elements

(LINE-1) is a well-established measure of DNA methylation often used
N27 dopaminergic cells

in epidemiologic studies. In a study to characterize the relationship of

age, sex, and prenatal exposure to persistent POPs, DDT, DDE and
cell line TK-6 was

PBDEs with DNA methylation in the birth cohort of Mexican-American

In vitro SC- M1
Model system

children participating in the CHAMACOS study. They measured the Alu

(HUVECs)
Zebrafish
Zebrafish
and LINE-1 methylation in whole blood sample of newborns and nine
Humans
Mice

Mice year old children. Their study suggested that repeat element methyla-
Rat
Rat

tion were only identified after examining the co-exposure of DDT/E

atherosclerotic vascular disease and oncogenesis

with PBDEs simultaneously. They also suggested that repeat element
Increase in inflammatory cytokines, cell cycle

methylation can be an informative marker of epigenetic differences by

age and sex and also prenatal exposure to POPs may be linked to hy-
pomethylation in fetal blood (Huen et al., 2014). Recently, microRNAs
Influence vascular inflammation and

are utilized as marker of pesticide exposure. MicroRNAs from the urine

Decrease in sperm concentration

May increase the risk of cancer

Decrease in lipid peroxidation

of 16 farm workers and 11 non-farm workers were isolated and iden-

tified using PCR arrays during two agricultural seasons. A total of 377
Transgenerational effect

microRNAs were detected and six were found to be positively asso-

arrest and apoptosis

ciated with farmworkers compared to non-farmworkers during the post-

harvest seasons. Five of these microRNAs trended positively with dose
Carcinogenesis

DNA damage

Tumorigenic
Cytotoxicity

response relationship with organophosphate metabolites in farm-

Apoptosis

Apoptosis

Toxicity
Toxicity

workers. Thus, microRNA can act as an early detection of pesticide

Effect

exposure (Weldon et al., 2016).

Histone H3, Histone deacetylase

6. Conclusions
Histone H3 and H4 acetylation

As research in the fields of pesticides has entered into the fifth

Global DNA methylation

CpG hypomethylatation
Epigenetic modification

expression
expression
expression
expression
Histone modifications
microRNA expression

microRNA expression

decade, the enormous information it has generated thus far serves as a

Genomic instability

DNA methylation

rich and instructive framework for the challenges and prospects that lie
CpG methylation
(HDAC) 4 and 7

forward. A combining concept is developing from the large number of

RNA
RNA
RNA
RNA

genetic, biochemical and cell biological studies are that the oncogenic
and other disease-causing potentials of pesticides demonstrate a dose
Micro
Micro
Micro
Micro

and context-dependent effects. It is evidenced that most of the pesti-

cides used worldwide can affect the normal cellular metabolism in one
Polychlorinated biphenyl

way or other. The major hurdle in such kind of studies is the limitation
Organophosphorus

of low dose chronic exposure to pesticides because animal models may

Organophosphate

Inorganic-arsenic
Organochlorine

Organochlorine

Organochlorine

Organochlorine

Organochlorine
Dicarboximides

Phenylpyrazole

show similar responses but on the other hand, the repair machinery
Organosulfur

associated with such responses is also different in these animal models

Carbamate

Conazoles

and humans, the life span and dietary pattern are also contrasting. For
Class

example, mice and rats have very fewer repair enzymes compared to
humans, which may affect the studies directly. For the sake of study, we
cannot voluntarily ask humans to feed upon pesticides containing diets.
Pesticides and associated epigenetic modulations.

Triadimefon, propiconazole, myclobutanil

These questions are challenging and new models are required to be

p,p′-DDT, its metabolite p,p′-DDE, and β-

Dichlorodiphenyltrichloroethane (DDT)

explored by the scientific community. Many studies have shown a sig-

nificant correlation with pesticides poisoning and oncogenic modula-
tions, additionally, there are studies which support these findings to a
lesser extent. At the molecular level, it is much established that pesti-
cides can cause oxidative stress and act as endocrine disruptors, and
Methyl isothiocyanate

their DNA and other macromolecule damaging properties can also

Hexachlorohexane
Sodium arsenite

modulate gene expression. Such changes are mostly harmful to cells and
Methoxychlor

Aroclor 1260

play a key role in the pathogenesis of multiple disorders including

Vinclozolin

Triazophos
Dichlorvos

Propoxur
Paraquat
Pesticide

cancer. Studies related to pesticides and epigenetics are recently been

Dieldrin

Fliponil

started and findings indicate that pesticides can cause numerous mod-
ulations in DNA methylation pattern, histone modification and also at
the level of non-coding RNAs. Finally, an alternative to chemical pes-
Table 1

Serial
No.

10.

11.
12.
13.
14.

ticides is a major thrust area which requires advance research to de-

1.
2.

3.

4.

5.

6.

7.

8.
9.

velop less harmful, more specific and more efficient pesticides which

111
A. Sabarwal et al.
can be made available for its mindful utilization.
Conflict of interest
The authors declares that there is no conflict of interest.
Acknowledgements
We acknowledge the financial support provided by Central
University of Gujarat, India for ongoing work related to pesticides and
cancer and UPE-2, UGC-RN and DST-PURSE, JNU, New Delhi. Akash
Sabarwal and Kunal Kumar are supported by a fellowship from
University Grant Commission and Department of Science & Technology,
New Delhi, India.
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