Molecular Insight on Arsenic Induced Carcinogenesis: Metabolism, Oxidative Stress, Genotoxicity, Signal Transduction

1. Molecular Insight on Arsenic Induced
Carcinogenesis: Metabolism, Oxidative
Stress, Genotoxicity, Signal Transduction

2. ARSENIC
 Arsenic (As) is a metalloid with atomic number
33, found in air, water, food and soil from natural
and anthropogenic sources.
 Exists in both organic and inorganic forms with -
3, 0, +3 and +5 oxidation state.
 Arsenic is highly toxic in its inorganic form.
 As(III) is more hazardous and carcinogenic than
As(V).
 Food contains both organic (mainly in sea foods)
and inorganic arsenical, whereas drinking water
contains mainly inorganic arsenic.
Why Arsenic?
 Recognized as Group 1 human carcinogen by the
International Agency for Research on Cancer
(IARC).
 About140 million people around the world are
exposed to carcinogenic level of arsenic through
drinking water, having concentration higher than
10µg/L (WHO provisional guideline value).
Figure: World Map showing risk of Arsenic contaminated
drinking water (Source: Schwarzenbach et al., 2010)

3. Molecular Mechanisms for Arsenic induced Carcinogenesis
• Arsenic is non-mutagenic human carcinogen that induces tumor through unknown
mechanism.
• The toxicity of arsenic is a function of its oxidation state and level of methylation
during metabolism.

4. Metabolism of Arsenic- Role in Carcinogenesis
Source: Minatel et al., 2018

5. Figure: Metabolic pathway of Inorganic Arsenic

6. OXIDATIVE STRESS
 Oxidative Stress is the most studied mechanism and believed to be responsible for inducing or
accelerating several mechanisms to initiate, promote and progress tumor formation.
 Arsenic induce over production of reactive oxygen species (ROS) and reactive nitrogen species
(RNS), responsible for oxidative stress in mammalian cells.
 Induces formation of superoxide anion (O2
-), hydrogen peroxide (H2O2), hydroxyl radical (OH·),
singlet oxygen (1O2), nitric oxide (NO) and peroxyl radical (ROO·).
 Exact mechanism for Arsenic induced ROS production is not known. However, several mechanisms
are proposed.
Figure. Mechanisms for generation of Arsenic induced ROS (Source: Hu et al., 2020).

7. As3+
Overproduction of ROS and RNS
OXIDATIVE STRESS
Oxidative
DNA damage
Inactivation of
DNA repair
enzymes
Disruption of
Signal
Transduction
Genotoxicity
 Impaired DNA Repair mechanism
 DNAAdducts
 Chromosomal aberrations; SCE; MN
 Compromised DNA Damage Response
Signaling
 Crosslink formation between DNA and
Protein
 Uncontrolled Cell
Proliferation
 Abrogated DNA Repair
Checkpoints
 Inflammation
 Apoptosis
CARCINOGENESIS
Figure: Role of Arsenic induced Oxidative stress in Carcinogenesis

8. GENOTOXICITY
 Is produced either by direct interaction of substances with DNA (DNA
reactive) or by indirect actions of substances that introduce errors in DNA.
 Major effects: chromosomal aberrations (CA), sister chromatid exchange
(SCE), micronuclei formation (MN), DNA strand break, deletion,
aneuploidy. Other effects: genomic instability, gene amplification.
 Mechanism: induction of oxidative stress or altered DNA repair machinery.
 Generation of ROS is considered to be main threat as it induces several DNA
damages (Figure).
Consequences of Arsenic Toxicity:
 Clastogenic (structural abnormalities) effect is observed at all doses of iAs
while aneuploidogenic (numerical abnormalities) effects are noted mainly at
higher doses of iAs.
 At high dose As also induces chromosomal erosion and pulverization.
 Arsenic binds with tubulin and inhibits microtubule assembly leading to
disruption of spindle formation.
 Most in vivo studies have shown positive genotoxic effect on humans. An
increasing incidence of bladder and lung cancer are observed in population
exposed to iAs. Increased incidences of CA, SCE and MN is also reported
from the human exposed to Arsenic through drinking water.
Figure. Schematic representation of arsenic
genotoxicity (Source: Faita et al., 2013).

9. INHIBITION OF DNA REPAIR MECHANISM
 Inorganic Arsenic and its trivalent methylated metabolites can
inhibit base excision repair (BER), nucleotide excision repair
(NER) and DNA Double-strand breaks (DSB) repair pathways.
 Inhibits DNA repair either down-regulating gene expression or
reducing the catalytic activities of DNA repair proteins or enzymes.
 DNA repair enzymes or proteins such as OGG1, DNA polymerase
β, APE1, DNA ligase I, XPB, XPC, XPD, XPF, ERCC1,
RFC,TP53, PCNA, are affected by As exposure.
 Alters DNA repair proteins function by binding directly and
strongly to 3 or more cysteine residues, replacing Zn2+ in the zinc
finger (ZnF) proteins like Xeroderma pigmentosum group A protein
(XPA) and poly(ADP-ribose) polymerase 1 (PARP-1).
 As induced ROS and RNS, impair enzymatic activity of ZnF via
oxidizing cysteines to form oxidization products, such as –SOH, –
SNO and −S−S− (Figure).
 As is found to induce DSBs and also inhibit DNA DSB repair by
either favoring error-prone NHEJ repair while inhibiting the error-
free HR pathway, ultimately contributing to genome instability.
Figure. Mode of action of Inorganic Arsenic to
inhibit enzymatic activity of DNA repair protein
(Source: Tam et al., 2020)

10. SIGNAL TRANSDUCTION PATHWAY
 Signal transduction is a process through which extracellular signals
is transmitted inside cell via intracellular series of signaling
molecules, to stimulate a cellular response.
 Arsenic alter signal transduction, via activating or inhibiting
regulatory proteins, transcription factors, which bind to DNA and
regulates gene transcription.
 As activates all three class of mitogen-activated protein kinases
(MAPKs)- p38 kinases, extracellular-regulated kinases (ERKs) and
the c-Jun N-terminal kinases (JNKs).
 As activates phosphorylation of epidermal growth factor receptor
(EGFR) by stimulating c-Src, and also stimulate its downstream
components like Ras, Raf, MEK, ERK, thereby resulting in cell
proliferation.
 As induced ROS activates p38 and JNK, which in turn activates
the transcription factor- activator protein-1 (AP-1), a heterodimer
protein belonging to family of proto-oncogenes, c-jun and c-fos,
thereby promoting tumor formation.
 As activated MAPKs pathway also affect Nuclear factor-κβ (NF-
κβ) activation, a dimeric transcription factors responsible for
regulating apoptosis, proliferation.
 As(III) decreases nuclear factor erythroid-2-related factor 2 (Nrf2)
level, and its downstream proteins, resulting in malignant
proliferation.
Source: Jomova et al., 2011

11. Figure. Arsenic trigger carcinogenesis via altering signaling pathways (Source: Flora, 2011).
• Arsenic induced ROS causes apoptosis directly or via the activation of p53 pathway. As exposure results in over expression of p53 protein, leading to DNA
damage & resulting in apoptosis. Additionally, ROS activates Iκβ complex (inactive NF-κβ) by phosphorylating by Iκβ kinase (modify thiol group in kinase).
This phosphorylation leads to the disassociation and degradation of Iκβ from active NF-κβ (p65 and p50), which activate various genes. Other molecules such
as p38, JNK, and ERK also activate NF-κβ. These factors along with others activate pro- or anti-inflammatory cytokines that could play important roles in
carcinogenesis.
• Effects of Arsenic on NF-κβ is cell-specific and dose-dependent. Low concentration (<5 µM) induce NF-κβ activation while high concentrations (>10 µM)
inhibits activation. As induced ROS is considered the main cause of NF-κβ activation at low dose.

12. CONCLUSION
 Arsenic is genotoxic non-mutagenic human carcinogen that induces tumor through unknown mechanism.
 Main molecular mechanism by which Arsenic induces tumor are oxidative stress, inhibition of DNA repair and
epigenetic modifications.
 Metabolism of arsenic involve both detoxification and bioactivation process, playing a crucial role in As
carcinogenesis both at genetic and epigenetic level. Biomethylation results in generation of ROS which plays a
crucial role to commence tumorigenesis.
 From genotoxicity studies, it can be believed that Arsenic facilitates the speedy transition of benign tumor to
malignant.
 A few follow up for arsenic research in future are:
• To find a suitable animal model for investigating arsenic-induced carcinogenesis mechanism in human
beings. Further, knowledge of mechanism would be helpful in understanding the carcinogenesis and to
investigate on its potential therapeutic side.
• To investigate more whether Arsenic effect DNA directly or indirectly.
• Differences in individual human susceptibility to arsenic should be further investigated to put more light on
arsenic induced carcinogenesis in human.
• To conduct risk assessment studies in populations exposed to low levels of arsenic so as to establish a
permissible limits of arsenic in drinking water.

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15. THANK YOU