article

1. Heavy metal and pesticide exposure: A mixture of
potential toxicity and carcinogenicity
David R. Wallace1,2
and Aleksandra Buha Djordjevic3
Abstract
There is a growing body of evidence that various pesticides
and heavy metals are carcinogenic. If not directly, there is also
evidence that shows that these compounds can participate in
carcinogenesis in a passive or permissive role, facilitating
other compounds from inducing tumor formation. Little evi-
dence is available to aid in understanding the toxicity of metal-
pesticide mixtures. In many instances, exposure to subclinical,
or subtoxic, levels would be asymptomatic under a single-
chemical exposure. But, we do not know how these com-
pounds would act together. A synergistic or potentiating
response could be highly possible. By chemically interacting
with the environment, as well as each other, metal pesticide
mixtures may yield unpredictable toxicity. Because we are not
exposed to a single xenobiotic at a time, the importance of
studying the toxicity of mixtures has never been more critical.
Addresses
1
Department of Pharmacology, School of Biomedical Science, Okla-
homa State University Center for Health Sciences, Tulsa, OK 74107-
1898, USA
2
Interdisciplinary Toxicology Program, Oklahoma State University,
Stillwater, OK, 74078-2003, USA
3
Department of Toxicology ‘Akademik Danilo Soldatovi
c’, Faculty of
Pharmacy, University of Belgrade, 11000, Belgrade, Serbia
Corresponding author: Wallace, David R. (david.wallace@okstate.
edu)
Current Opinion in Toxicology 2020, 19:72–79
This review comes from a themed issue on Mechanistic Toxicology
Edited by Aleksandra Buha Djordjevic, Jonathan Powell, Aristides
Tsatsakis and David Wallace
Available online 15 January 2020
For a complete overview see the Issue and the Editorial
https://doi.org/10.1016/j.cotox.2020.01.001
2468-2020/© 2020 Elsevier B.V. All rights reserved.
Keywords
Cadmium, Mercury, Arsenic, Nickel, Organophosphate, Organochlo-
rine, Carbamate, Pyrethroid, Neonicotinoid.
Abbreviations
AChE, acetylcholinesterase; As, Arsenic; CAR, carbamate; Cd, cad-
mium; Cr, chromium; DDE, dichlorodiphenyldichloroethane; DDT, 4,40-
dichlorodiphenyltrichloroethane; ER, estrogen receptor; FSH, follicle-
stimulating hormone; IARC, International Agency for Research on
Cancer; IGF-1, insulin-like growth factor-1; LH, luteinizing hormone; Pb,
lead; Hg, mercury; MUC-1, mucin-1; Ni, nickel; Nrf2, nuclear factor
erythroid 2–related factor 2; OC, organochlorine; OP, organophos-
phate; PYR, pyrethroid.
Introduction
We are blindly exposed to multiple toxicants daily.
Concern over the potential danger from exposure to
heavy metals and pesticides has grown owing to their
ubiquitous nature. We will examine the foundational
toxic mechanisms by which pesticides and heavy metals
may be carcinogenic and then address the growing
concern for the toxicity of chemical mixtures. An enor-
mous body of evidence points to environmental factors
as one of the foundations of cancer development.
Governing bodies which have regulatory oversight have
developed classifications for carcinogens (Table 1). The
terminology is vague, and there is overlap between the
different classifications [1,2]. Our lack of foundational
knowledge regarding the toxic actions of pesticides and
metals in humans, not only individually, but in mixtures
has led to confusion with study outcomes, data inter-
pretation and finally finding a true classification for the
actions of pesticide-metal mixtures. Another reason that
the study of chemical mixture toxicity is essential is that
the individual chemical may not be an actual carcinogen
alone but instead, is a ‘cocarcinogen.’ A cocarcinogen is not
carcinogenic alone, but in the presence of a second
compound, will facilitate carcinogenesis. This facilita-
tion can also be called passive or permissive toxicity.
Individual pesticide toxicity
For nearly a half century, there has been evidence
supporting the carcinogenicity of various pesticides [1].
A review by Sabarwal et al. [3] has pointed to several
mechanisms that are involved in pesticide carcinoge-
nicity including DNA damage, oxidative stress, and
epigenetic changes. We will consider the broad classes of
pesticides and examine individual pesticides for unique
characteristics. Some of the leading pesticides are listed
in Table 2 with their International Agency for Research
on Cancer categorization. A general depiction of po-
tential pesticide activity leading to tumor formation is
shown in Figure 1b.(See Figure 2)
Organophosphates
Although considered weak carcinogens, exposure to or-
ganophosphates (OPs) such as malathion, are signifi-
cantly associated with the development of non-Hodgkin
lymphoma [4,5]. Inhalation of OPs can lead to cellular
death by increased oxidative stress, disruption of mito-
chondrial function, and upregulation of the executioner
caspase, caspase-3 [6].
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2. Organochlorine
The carcinogenic effects associated with organochlorine
(OC) exposure include the generation of free radicals,
impairment of antioxidant responses, decrease in
executioner caspase activity (caspase 3 and 7), and
alteration of mitochondrial membrane potential [7].
4,40-dichlorodiphenyltrichloroethane and other OCs
linked with a positive association with breast cancer
incidence are methoxychlor, chlordane, pp’-dichlor-
odiphenyldichloroethane and polychlorinated
biphenyls-52 [8,9].
Carbamate
Pesticides have been shown to the function of cell
mitochondria and induce apoptosis in Tcells, leading to
tumor development [10]. Findings of altered mito-
chondrial function and T cell activity may explain the
incidence of immunotoxicity and carcinogenicity
attributed to carbamate (CAR) after long-term exposure
[10]. CAR exposure can increase the immune response,
increase oxidative stress, alter immune and hormonal
responses, ultimately leading to tumor formation [11].
Extending cellular in vitro CAR studies, human cohort
studies have substantiated the hypothesis that CAR
exposure can lead to tumor formation in the central
nervous system [12].
Pyrethroid
The EPA lists cypermethrin (type II pyrethroid [PYR])
as ‘possible’ carcinogen, whereas other type I and type
II PYR agents are considered ‘not likely’ to be carci-
nogenic [1,13]. Cypermethrin promotes macrophage-
induced tumor metastasis in the lung and is signifi-
cantly more toxic to astrocytes than other PYR com-
pounds [14]. More work is needed to better
understand the toxicity of PYR compounds and their
carcinogenic properties.
Neonicotinoid
Reported neonicotinoid toxicity includes increased
oxidative stress, leading to cellular damage and the
generation of toxic metabolites [15]. Neonicotinoids
have been shown to upregulate the expression of
CYP3A7, resulting in increased enzyme activity [16].
Table 1 Carcinogenicity categories by organization.
IARC Group No. GHS Category No. NTP ACGIH EU Category No.
1 [definite] 1A [known – human studies] Known A1 [confirmed] 1 [known]
2A [probably] 1B [known – animal studies] Suspected (likely) A2 [suspected] 2 [probably]
2B [possibly] 2 [suspected] A3 [confirmed in animals – unknown in humans 3 [possible]
3 [not classified] A4 [not classified]
4 [not carcinogen] A5 [not carcinogen]
IARC = International Agency for Research on Cancer; GHS = Globally Harmonized System; NTP = National Toxicology Program; ACGIH = American
Conference of Governmental Industrial Hygienists; EU = European Union.
Table 2 Classification of pesticides and heavy metals by IARC.
Pesticides Heavy metals
Organophosphate Organochlorine Carbamate Pyrethroid Neonicotinoid
Parathion [2B] DDT*
[2A] Aldicarb [3] Permethrin [3] Imidacloprid Nickel [1, 2B]
Cadmium [1]
Chromium (VI) [1]
Chromium (III) [3]
Inorganic mercury [3]
Organic mercury [2B]
Inorganic lead [2A]
Organic lead [3]
Lead [2B]
Arsenic/inorganic [1]
Organic arsenic [3]
Nonarsenicals [2A]
Malathion [2A] Dieldrin [2A] Carbaryl [3] Resmethrin Thiamethoxam
Chlorpyrifos Lindane [1] Propoxur Phenothrin Clothianidin
Numbers in [] represent International Agency for Research on Cancer (IARC) ratings – [1] = Carcinogenic to humans; [2A] = Probable human carcinogen;
[2B] = Possible human carcinogen [3]; = not currently classified as a carcinogen. No rating means the compound has not been reviewed, or is undergoing
additional evaluation.
*
DDT = 4,40
-dichlorodiphenyltrichloroethane.
Carcinogenicity of pesticide-metal mixtures Wallace and Buha Djordjevic 73
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3. Increased CYP3A7 activity alters the hydroxylation of
dehydroepiandrosterone, which is a source of estradiol.
Individual metal toxicity
Metals are mainly found as mixtures in various parts
of the ecosystem and can interact with other com-
pounds, changing the toxicokinetic and toxicodynamic
profiles for each compound. In many instances, tumor
formation is a physiological response (Table 2). As
additional emphasis is placed on elucidating the
pathways associated with the carcinogenic and muta-
genic effects of metals, researchers have tried to
outline the various mechanisms of metal-induced
carcinogenesis [17,18]. A general schematic of
metal-related effects resulting in tumor formation is
depicted in Figure 1b.
Lead
The International Agency for Research on Cancer clas-
sifies lead (Pb) as group 2B (possible carcinogen) and
inorganic Pb as group 2A (probable) carcinogen [19].
Pb-induced carcinogenicity is owing to increased
oxidative stress, membrane alterations, impaired cell
signaling, and neurotransmission [20]. Evidence
describing direct genotoxic actions of Pb in humans is
lacking, but indirect genotoxicity may be possible
through increased oxidative stress and reduced DNA
repair [19].
Cadmium
Cadmium (Cd) is a recognized carcinogen [21] and is a
metal commonly found in the environmental, either
naturally, or through manufacturing processes. Cd is
highly persistent in the body, and the environment and
this persistence has led to an increased health risk [22].
We have reported that pancreatic tumors exhibit higher
levels of Cd than surrounding or normal tissue and may
exert a fraction of this toxicity by altering mitochondrial
function [23,24].
Mercury
Exposure elicits the characteristic cellular responses of
increased oxidative stress, decreased DNA repair,
increased production/release of proinflammatory cyto-
kines, and altered membrane permeability as a means
of inducing carcinogenesis [25]. Existing data has not
associated mercury (Hg) exposure with tumor forma-
tion in humans, with only a small body of work in
animals indicating Hg-related carcinogenicity [26].
Humans exposed to Hg have demonstrated genotoxic
changes measured using micronucleus assays and
comet formations [27]. Other investigators have sug-
gested that Hg functions more as a ‘promoter’ of
tumor formation by altering downstream methylation
[28].
Chromium
Unlike other metals, chromium (Cr) appears to exert its
carcinogenic effects via mutagenesis [29]. In vitro
studies using lung epithelial cells, Cr exposure was
shown to elevate oxidative stress involving Nrf2 and
altered expression of antioxidant proteins before cell
transformation [30,31]. Mixtures of Cr and other metal
species is an important area of study to better under-
stand the cellular mechanisms leading to tumor
formation.
Figure 1
Schematic of potential mechanisms for pesticide and metal carcinogenicity. (a) Two mechanisms associated with pesticide damage to cellular function are
through either direct interaction with DNA or epigenetic changes. These genetically related changes alter normal cellular function, promoting tumor
formation. (b) Metals can act through multiple pathways involving oxidative stress and an increased generation of free radicals. Elevated free radical
content leads to protein oxidation, lipid peroxidation, or direct damage to DNA. Cellular alterations following oxidative damage can lead to tumor formation.
74 Mechanistic toxicology
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4. Arsenic
Exposure has been found to be carcinogenic at As con-
centrations that are at or below the As reference dose
[32]. Inorganic As is metabolized to the trivalent form
and has actions similar to Hg in that As will bind to
sulfhydryl groups of proteins but does not bind to DNA
[33]. There have also been reports of increased oxida-
tive stress and the facilitation of DNA damage [34]. An
intriguing response to As exposure is the biphasic
response of the apoptotic PI3K/AKT/mTOR pathway
after exposure to As, normal growth is increased, but
cancerous growth is suppressed [35]. Chronic exposure
to Al has been associated with behavioral and neuro-
logical changes [36].
Toxicity after exposure to metals is dependent on (1)
species of the metal to which one is exposed, (2)
duration of the exposure, (3) route of exposure and (4)
organ system being investigated. The need to control
each of these variables has led to a wide range of reports
on metal responses, from no effects to toxic effects to
even beneficial effects. Not entirely surprising because
there are metal-based therapeutics that is currently
used today, such as platinum-based drugs used in
chemotherapy.
Potential pesticide–metal interactions
within the soil
To date, there have been few investigations into the
toxic effects of environmental metalepesticide mix-
tures on humans. Environmental exposure brings in a
complicating factor, the presence of the humates. Early
studies have already demonstrated an interaction be-
tween gamma-hexachlorocyclohexane (LindaneÔ), a
variety of heavy metals and humate [37]. Another early
study demonstrated that glyphosate will strongly com-
plex with an ironehumic acid complex to form a bigger
complex [38]. Recently, investigators have reported that
CAR pesticides bind with higher affinity to both humic
and fulvic acid than metals and can thus displace the
metals back into the environment [39]. Examining the
metalehumic acid interaction, with both divalent and
trivalent metals complexing with humic acid, the
strongest metalehumic acid complexes were formed by
iron and lead [40]. Interactions between pesticides,
metals, humic acid, and fulvic acid involve highly com-
plex chemical reactions. An area that is not heavily
investigated, the potential chemical interactions be-
tween chemicals such as pesticides and heavy metals
and their ability to alter chemical responses is a subject
that needs more scrutiny and attention. The foundation
for what nonhuman and human organisms will be
exposed begins with modifications within the soil and
sediment.
Toxicity of metal–pesticide mixtures:
nonhuman
There is a growing interest in the study of chemical
mixture toxicity and increasing our understanding of
the coexposure effects [41]. Exposure in an aquatic
environment has been a likely site to investigate
because most metals and pesticides will eventually find
their way into the aquatic environment. Exposure to
metalepesticide combinations has yielded mixed re-
sults. Using the zebrafish (Danio rerio) as a model
system, both synergistic and antagonistic results have
been reported which were dependent on the method-
ology of the study. The combination of buprofezin
Figure 2
Venn diagram schematic which highlights the individual toxicity of pesticides and metals alone, and the potentially unique toxicity associated with the
mixtures, as well as the individual pesticide and metal toxicity that contribute to the overall toxicity of pesticide–metal mixtures.
Carcinogenicity of pesticide-metal mixtures Wallace and Buha Djordjevic 75
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5. (homopteran inhibitor of chitin biosynthesis) and Ni
resulted in a very robust damage of zebrafish embryos
via elevated oxidative stress, and an increased toxicity
owing to the buprofezineNi complex formed [42]. The
buprofezineNi complex facilitated the transport of
nickel into the embryo. A combination of Cd and Ni in
the presence of deltamethrin reduced deltamethrin-
associated behavioral toxicity as indicated by the
maintenance of swim behavior compared with the
reduction observed in the presence of deltamethrin
alone [43]. The authors speculated that this was due to
a CdeNi interference in deltamethrin reduction of
antioxidant enzymes. The combination of glyphosate
and arsenic significantly altered tadpole (Rhinella
arenarum) development by increasing levels of oxidative
stress, increasing thyroid hormone levels, and frag-
mentation of DNA [44].
In the earthworm (Eisenia fetida), the combination of
pesticide and heavy metals is critical for assessing
damage. The combination of the urea-based herbicide,
Siduron, with Cd displayed a synergistic toxicity in the
earthworm with a significant increase in the lethality
[45]. Yet, a Cd atrazine combination was only weakly
toxic, whereas a combination of Cd and chlorpyrifos
were highly toxic to the earthworm [46]. Rai et al. [47]
demonstrated that mixtures of CAR pesticides signifi-
cantly reduced high-density lipoprotein and increased
other lipids to an extent greater than after exposure to
individual pesticides. Similar results were seen in
plants exposed to heavy metals. The combination of
lead and zinc synergistically increased the amount of
zinc uptake into the leaves, but was antagonistic in the
root system, decreasing zinc uptake [48]. A recent
review [49] found that in most instances, the combi-
nation of pesticide and metal effects were synergistic,
compared with the toxic effects of individual com-
pounds. More elaborate analysis in earthworms using
Cd as the metal with four pesticides, used various
mixtures of Cd with the pesticides to provide infor-
mation up to mixtures containing four different
chemicals [50]. Using concentrations of each com-
pound below their toxic threshold, the investigators
reported an increase in oxidative stress, glutathione
depletion, and lipid peroxidation that was dependent
on the combination of compounds. The primary
conclusion from these studies was that the reported
effects occurred at concentrations that would have
usually been subtoxic for each of the compounds alone,
suggesting a synergistic or potentiating effect of the
combination of metals and pesticides.
Collectively, from the nonhuman studies we have been
able to see that exposure to combinations of pesticides
and heavy metals are more toxic than exposure to in-
dividual chemicals. Whether this effect is additive,
potentiating or synergistic is still open for discussion,
but in most instances, the results were more than
additive. A key consideration for these studies is that
most used concentrations of the pesticide or heavy
metal that was relevant to what was measured in the
environment.
Toxicity of metal–pesticide mixtures:
human
The importance of studies examining the effects of
chemical mixtures on human has been stressed in
three recent reviews [41,51,52]. A recent study
examining the concentrations of 16 metals and 6 pes-
ticides found that there were seasonal changes in the
levels of each compound identified [53]. The combi-
nation of fenitrothion and Cr increased the severity of
testicular toxicity [54], possibly through an increase in
oxidative stress, reduction in free radical scavenging
capacity, and androgenic hormone changes. The com-
bination of Cd and chlorpyrifos increased hepatic
toxicity greater than the sum of each compound alone
through a synergistic toxicity evident in the increased
levels of cholesterol and triglyceride accumulation in
the cells [55,56]. Chen et al. [56] demonstrated that
Cd and chlorpyrifos form a complex which facilitated
the cellular entry of each chemical, speculating that
this complex acted as a Trojan horse facilitating entry
of both Cd and chlorpyrifos resulting in augmented
cellular toxicity. One indirect measurement study in
humans examined the concentrations of pesticides and
metals from toenail clippings and their conclusion was
exposure to pesticides increased the uptake of several
metals, with Cd being the primary metal, increasing
the likelihood of toxicity [57]. The ability of a pesti-
cide to facilitate metal uptake implies a passive or
permissive interaction between the two compounds.
The major interest of our group is the study of envi-
ronmental factors and the development of pancreatic
cancer. Other investigators have shown significant as-
sociations between pancreas exposure to pesticides
and heavy metals, Cd in particular [58], whereas the
metals, nickel, and Cr were not significantly associated
with pancreatic cancer [59]. Recently, it was postu-
lated that mixtures of chemicals resulting in synergis-
tic effects only occurred at high concentration, and
instead, we should be concern with the cumulative
effects of exposure to chemical mixtures such as pes-
ticides and heavy metals [60]. Collectively, there is an
increasing body of evidence that supports the associ-
ation between pesticides and heavy metals with the
development of cancer.
Summary and conclusions
It is clear from the information presented here and the
previous data from toxicology studies years ago, expo-
sure to pesticides and/or heavy metals can lead to
adverse consequences regarding health. Whether there
is direct toxicity or a passive, or permissive, toxicity, the
ability of combinations or mixtures of chemicals to
76 Mechanistic toxicology
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6. elicit toxicity is enhanced. Our understanding of the
mechanistic toxicity of pesticides and metals is limited.
In many instances, we know little about the cellular/
molecular changes that are occurring. The toxicity of
the mixtures is the ‘toxicological abyss,’ where our
understanding of potential chemical and cellular in-
teractions, the ability of compounds to potentiate or
synergize with the other compound, is a large void. We
can hypothesize about potential interactions. For
example, in the soil, chemicals may bind to humic or
fulvic acid essentially inactivating them. But, another
chemical which binds stronger, displays the first
chemical from its humic/fulvic bond releasing the toxic
compound into the environmental. The binding to
humic and fulvic acid can also result in what can be
thought of as a depot for toxic chemicals. Another area
would be the study of the how humic or fulvic acid may
change the chemical composition or activity of the
parent chemical, leading to elevated toxicity. The
ability of various pesticides and heavy metals to form
complexes as added another layer of toxic complexity. A
metal that may not normally be able to enter a cell
unless extremely high concentrations are present, will
complex with a pesticide, acting as a Trojan horse to get
that metal into the cells in high enough concentrations
that toxicity is observed. Overall, a significant effort
needs to be made to study the fundamental effects of
chemical mixtures and then to develop algorithms that
can be used as predictive models for more complex
mixtures. It is a daunting task, and at times may seem
like catching water with a sieve, but a systemic
approach needs to be developed to study these myriad
of possibilities.
Acknowledgments
The authors would like to acknowledge that the image used in the
graphical abstract was obtained and reproduced with permission from
Singh et al. [49].
Author Contribution
David Wallace: Conceptualization, Supervision, Writing
- Original Draft, Writing - Review Editing; Aleksandra
Buha Djordjevic: Conceptualization, Writing - Original
Draft, Writing - Review Editing
Funding
The authors received support in part from OSU intra-
mural funding [#154333 #154357] and Ministry of
Education, Science and Technological Development of
Serbia (Project III 46009). The funders had no role in
the writing of the manuscript.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could
have appeared to influence the work reported in this
paper.
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