Download Arsenic-Induces Dysfunction in Poly (ADP-Ribose)

Arsenic-Induces Dysfunction in Poly(ADP-Ribose) Polymerase-1:
Arsenic Contamination in Potable Water and an Assessment of Regulations
California State University Northridge; Department of Environmental and Occupational Health
Macario Perez, Ammar Witwit and Antonio F. Machado
Abstract
Biotransformation
Health Effects
Arsenic in potable water supplies one of the greatest public health concerns Human exposure to iAs via drinking water is converted to trivalent and
worldwide. Negative health effects from exposure to arsenic in drinking water pentavalent methylated metabolites, monomethylarsonous acid (MMAIII) and
have been recognized for decades. In 2001, the USEPA adopted a new
dimethylarsinous acid (DMAIII), monomethylarsonic acid (MMAV) and
maximum contaminant level (MCL) for arsenic in drinking water. The new dimethylarsinic acid (DMAV) [9]. MMAIII and DMAIII are the bioactivated
standard was set to ten parts per billion, replacing the old standard of fifty
intermediate metabolites formed during metabolism. Monomethylarsonic acid
parts per billion. At the time, the agency established the updated standard to (MMAV) and dimethylarsinic acid (DMAV) are the stable methylated
protect public health by utilizing the best available science and technology as metabolites formed at the end of biotransformation and are taken up by
well as considering feasibility. Much has changed since the update.
phosphate transporters and excreted via urine [10].
Historically, arsenic standards have been set because of its carcinogenic
The AsIII and AsV species are quickly and extensively absorbed in the
ability. The available science of today shifts the focus away from solely
gastrointestinal tract [3]. The biotransformation of the metalloid follows a
addressing the carcinogenic potential of arsenic and evaluates the cosequential process and is characterized by two main reactions (Figure 1). The
carcinogenic potential of the metalloid by studying the effect of arsenic on
iAs compounds undergo reduction reactions from the AsV to AsIII oxidative
functional genomic stability proteins such as poly (ADP-ribose) polymerasespecies, followed by an oxidative methylation reactions in which the AsIII
1. Research of today also focuses on evaluating the association between
oxidation state of iAs is sequentially methylated to form the monomethylated
arsenic exposure and non-carcinogenic diseases. The data of today confirms
and dimethylated products utilizing S-adenosylmethionine (SAM) as the
the current drinking water standard does not adequately protect public health
methyl donor and glutathione (GSH) as an essential co-factor [3,9]. The initial
by revealing synergistic carcinogenicity associated with iAs exposure by
reduction reaction can occur enzymatically or non-enzymatically in the
inhibition of genomic repair mechanisms along with decreased toxicological
presence of a thiol such as GSH [9]. Arsenic methyltransferase (AS3MT), also
endpoints associated with non-carcinogenic diseases such as diabetes
called arsenite methyltransferase, is a key enzyme in the metabolic pathway of
mellitus.
iAs after the initial reduction step. AS3MT catalyzes the transfer of a methyl
group from SAM to AsIII species, resulting in the production of the methylated
metabolites [11].
Introduction
Arsenic (As) is a naturally occurring ubiquitous element, classified as a
metalloid. In the environment, arsenic can combine with oxygen, chlorine
and sulfur to form inorganic arsenic compounds (iAs). Arsenic in animals
and plants combines with carbon and hydrogen to form organic arsenic
compounds. The iAs species have been associated with long-term health
effects and are of special concern due to their presence in potable water
sources worldwide. Exposure to iAs in potable water sources is considered
one of the most significant public health concerns worldwide. Hundreds of
millions of people worldwide are exposed to high levels of iAs via drinking
water. Since 1997, As has been the number one substance in the
Comprehensive, Environmental, Response, Compensation and Liability Act
(CERCLA) Priority List of Hazardous Substances published by the Agency
for Toxic Substances and Disease Registry [1]. The substances on this
priority list are ranked on frequency or occurrence, toxicity and potential for
human exposure.
Production, Use, Fate & Transport
The iAs species form naturally from the earth’s crust and also from
industrial activities, which contribute to the overall contamination level in
drinking water sources. Geological factors significantly affect the levels of
As in ground water supplies. For example, Bangladesh is one of the most
severely impacted regions for As contamination in potable water, due to
the Himalayan mountains emitting the metalloid into rivers that
contaminate drinking water sources [2]. The compounds also arise as a
byproduct from the smelting of copper, lead, cobalt and gold ores. The
smelting of non-ferrous metals and the production of energy from fossil
fuels are the two major industrial processes that lead to anthropogenic As
contamination in water [3]. The United States (US) is the world’s leading
consumer of As, with the major use being in wood preservation utilizing
copper chrome arsenate (CCA)[4]. Other industrial uses where iAs
compounds get contributed into potable water sources include the
production of agricultural chemicals, as an alloying element in ammunition
and solders, as an anti-friction additive to metals used for bearings and to
strengthen lead acid storage battery grids. These metal-mining operations
are also significant contributors to the overall contamination levels in
potable water, due to leaching in soil from surrounding areas [5]. High
purity As is used by the electronics industry for gallium-arsenide
semiconductors in telecommunications, solar cells and space research.
Various organic arsenicals are utilized in the US as herbicides and as
antimicrobial additives for animal and poultry feed [5].
The presence of iAs in potable water sources and other parts of the
environment stems from the biogeochemical cycle of the metalloid, which
explains the mobility of the metalloid from the solid state to the aqueous
state [6]. Microbial mobilization of As into the aqueous phase is one of the
main mechanisms spreading iAs contamination in drinking water systems
[6]. These microbes have evolved over time via horizontal gene transfer
(HGT) to develop As-resistance, which affects the overall As speciation
and mobility in the environment [6]. Microbial influences affecting the
geochemistry include catalyzing redox transformation and other reaction
that affect how As travels in subsurface environments. Chemical speciation
of As is related to the oxidation of As [7]. Abiotic and biotic mechanisms
have also been hypothesized to explain subsurface mobilization of As in
groundwater supplies such as reductive dissolution of As-rich Feoxyhydroxides, oxidation of As-rich pyrite and weathering of minerals that
contain either phosphate, ammonia or iron [7].
Chemical speciation of As is related to the oxidation state in which the
compound is in, which is influenced by environmental factors such as pH
[3,7]. The two common oxidative states of iAs humans are exposed to via
drinking water are the pentavalent and trivalent forms, known as Arsenate
(AsV) and Arsenite (AsIII) [3]. AsV and AsIII are the anionic forms of
arsenic acid and arsenous acid. These two oxidation states of iAs regulate
the mobility and toxic potential of the metalloid [7]. Both have differing
toxicological effects as well as differing environmental impacts. AsV is the
predominate form of iAs in oxic subsurface systems and is less harmful to
the environment because the oxidative state enables the metalloid to be
favorably adsorbed onto solids particles. AsIII is the predominate form in
anoxic environments and does not adsorb onto solid particles as well as
AsV, making it more mobile in the environment [7].
Levels in Environment
There are believed to be more than thirteen million people within the US
exposed to drinking water levels exceeding the federal maximum
contaminant level (MCL) of 10 parts per billion (ppb) [2]. Levels of As
found in ground water systems, which small communities typically rely on
for drinking water, tend to be greater than those found in surface water
sources such as lakes and rivers, which larger cities rely upon [5]. The As
species found in potable water is almost entirely in the iAs form. Due to
the many factors influencing the overall iAs contamination levels, the
level of contamination in potable water sources is unequally distributed
across the US. Western states have more water systems with levels
exceeding the MCL, with some exceeding 50 ppb. Parts of the Midwest
and New England also have some water systems with iAs levels exceeding
10 ppb, but most systems meet the current standard [8].
Since the current federal MCL for iAs was set over a decade ago, numerous
studies have revealed the standard to be inadequate at protecting against
long-term health effects. Numerous epidemiological and animal studies have
shown a clear dose-dependent relationship to chronic iAs exposures along
with their methylated metabolites to be associated with increasing the
incidence of serious diseases such as various cancers, liver injury,
neurotoxicity, peripheral vascular disease and endocrine dysfunction
[12,13,14,15].
The adverse health effects associated with high levels of iAs exposure via
drinking water have been understood for decades. Both the AsIII and AsV
parent compounds and methylated metabolites pose some type of adverse
health related ability, with the trivalent species historically being the most
negative due to the generation of reactive oxygen species (ROS) and
reactive nitrogen species (RNS) [15]. Many factors will determine the level
of toxicity upon exposure to iAs such as the dose, duration of exposure and
the enzymatic ability of organisms to adequately metabolize and excrete the
metalloid.
The increased incidence of non-carcinogenic diseases along with the cocarcinogenic potential of iAs species are the two most significant findings
over the last decade. Taiwan and Bangladesh are areas with high levels of
iAs in potable water sources; as a result, a significant dose-response
relationship has been made between iAs exposure and the prevalence of
non-cancerous diseases such as diabetes mellitus stemming from oxidative
The methylation reaction, which is a phase II biotransformation reaction,
stress [12]. In vitro and in vivo experiments on rat pancreatic β-cell lines
serves the purpose of increasing the hydrophilic of a compound in order to
facilitate excretion. The metabolism of iAs compounds significantly influences (RIN-m5F) have indicated that iAs triggers oxidative stress to suppress
the extent of potential toxicity stemming from exposure to the metalloid due to insulin secretion in pancreatic β-cells. These experiments have also shown
the reactive AsIII intermediate metabolites in the process. Mammalian species iAs to induce pancreatic β-cell apoptosis through mitogen-activated protein
methylate iAs, with little variation between human populations in the rate and kinases (MAPKs) and mitochondrial dysfunction leading to the activation of
poly (ADP-ribose) polymerase-1 (PARP-1) and caspase cascades-meditated
extent of methylation. Factors such as dose, age, gender and smoking
signaling pathway [12](Figure 2). MAPKs regulate cell proliferation, gene
contribute very marginally to the variation in methylation rate amongst
expression, differentiation, mitosis, cell survival and apoptosis. In vivo
humans [3]. Biomethylation was believed to be a detoxification pathway for
experiments conducted on intact isolated adult male mice pancreatic islet
iAs. However, there is strong evidence from research towards adverse health
cells (C57BL/6) revealed that AsIII species were potent inhibitors of glucoserelated effects in humans and animal models chronically exposed or acutely
stimulated insulin secretion (GSIS) with MMAIII and DMAIII being the most
exposed to iAs, stemming from the bioactivated trivalent intermediate
potent insulin inhibitors [16](Figure 2).
metabolites. Over the last decade, data has shown bioactivated intermediates
to be as toxic or even more toxic than the parent iAs compounds [11].
The co-carcinogenic potential of iAs species has been investigated by
examining interactions with functional genomic stability proteins. Exposure
OH
OH
to iAs has been shown to enhance the persistence of deoxyribonucleic acid
GSH
SAM
(DNA) damage induced by ultraviolet (UV) light, poly-aromatic
O
As
OH
As
AS3M
T
hydrocarbons (PAHs), x-rays, alkylating agents and DNA crosslinking
OH
OH OH
OH
agents. Research has shown that the inhibition of DNA repair mechanisms
III
V
(2)
As
(1) As
O
As
CH3
via iAs exposure at micromolar concentrations amplifies the carcinogenic
GSSG
SAH
ability of other DNA damaging agents in the environment by causing
OH
inhibitory affects amongst critical DNA repair mechanisms (Figure 3)
V
OH
(3)
MMA
GSH
[17,18,19,20,21]. PARP-1 is a critical DNA repair enzyme, utilized by
As
numerous DNA repair mechanisms including the most widely utilized DNA
repair pathways, base excision repair (BER), nucleotide excision repair
CH3 CH3
GSSG
(NER) and double-strand break repair (DSBR). Arsenic-induced DNA repair
CH3
GSH
(6) DMAIII
CH3
inhibition has been extensively reported with PARP-1 used in BER and
SAM
AS3MT
As
O
As
CH3
NER [19]. PARP-1 is activated as an immediate cellular response to induced
OH OH
DNA damage by ROS or environmental agents. PARP-1 contains three
OH
(4) MMAIII
functional domains within its unique regulatory protein structure. PARP-1
V
(5) DMA
GSSG
SAH
contains an amino-terminal DNA-binding domain with two zinc fingers that
Figure 1: Biotransformation begins at (1) with the AsV form of iAs species; can also begin at (2). GSH reduces AsV by
V
III
removing an oxygen atom found in the As species to form the unstable As species (2) and glutathione disulfide
are critical for the binding of PARP-1 to single-strand and double-strand
(GSSG). In the next step, AS3MT catalyzes the methylation of AsIII from a methyl group found on SAM to form the
break sites. A third zinc finger is utilized for coupling damage-induced
stable monomethylated product, MMAV(3) and S-adenosylhomocysteine (SAH). The MMAV metabolite at this stage
may either be taken up by phosphate transporters and excreted via urine or can undergo another reduction reaction.
changes in the DNA-binding domain to the catalytic domain leading to
GSH can reduce MMAV to form another unstable trivalent compound, MMAIII(4) and GSSG. The unstable MMAIII is
alterations in PARP-1 activity [21]. Exposure to the parent iAs compounds
methylated by SAM using AS3MT to form another stable pentavalent metabolite, DMAV(5) and SAH. DMAV at this
point may be taken up by phosphate transporters and excreted or undergo another reduction reaction by GSH to form
and the bioactivated metabolites have been strongly shown to cause
another unstable trivalent compound, DMAIII(6) and GSSG.
dysfunction in PARP-1 by altering the zinc domains of PARP-1, which
B1
A1
provides strong evidence of the synergistic co-carcinogenicity due to
C1
E1
inhibition [19,20,21].
A4
B2
B3
B4
A2
C2
B5
A3
D1
Figure 2: The graphs labeled A1 and A-2 are derived from in vitro tests on RIN-m5F cells [12]. In A1, the cells
were pre-treated treated with arsenic trioxide (ATO) before undergoing an MTT assay to determine cell viability.
The β-pancreatic cells (RIN-m5F) decreased in viability in a concentration dependent manner, with the most
significant decrease observed in the lower doses. A2 from the same experiment shows insulin secretion from the
cells decreasing in a concentration dependent fashion. A3 shows results from an oral glucose tolerance test being
conducted in vivo from the same experiment [12]. The results showed plasma glucose levels to remain evaluated
in the presence of ATO, indicating cellular dysfunction in normal male ICR mice. A4 is a general schematic of
the pathways activated from ROS generation leading to cellular apoptosis [12]. The data provided in the Blabeled diagrams of this figure show how trivalent metabolites inhibit insulin-dependent phosphorylation of
protein kinases B (PKB/Akt) by pyruvate dehydrogenase kinase (PDK) [16]. It also proves iAs can not only
contribute to type II diabetes insulin resistant diabetes, but can also contribute to non-insulin dependent type II
diabetes [16]. B1 shows the inhibition of GSIS by trivalent arsenicals. Insulin secretion in C57BL/6 pancreatic
cells decreased in a concentration dependent fashion, with DMAIII being the most potent inhibitor and the
greatest inhibition clearly seen at the lowest dose [16]. B2 shows the content of insulin in control and treated
cells compared to levels secreted, again with DMAIII being the most potent inhibitor. B3 shows the relative gene
levels of Ins1 and Ins2 genes in control and treated cells, indicating genomic damage due to altered protein levels
and gene expression [16]. B4 shows an MTT assay with treated cells; authors concluded cells did not decrease in
viability at the levels of trivalent iAs species used and GSIS impairment was not related to loss of β-cell integrity,
suggesting inhibition is due to trivalent iAs interfering with the mechanisms involved with insulin transport [16].
Lastly, B5 shows the effect of trivalent arsenicals on insulin secretion levels in the presence and absence of an
anti-oxidant (KCL) [16].B5 reveals the insulin suppression ability stemming from ROS species at low levels.
F1
E2
Figure 3: The implications of iAs exposure on critical DNA repair pathways. Figure F1 is a 3-dimensional crystal
structure of the PARP-1 binding domain in complex with DNA [22]. The C1 and C2 graphs were the results provided by
researchers conducting an experiment on the interaction between iAs species an Fanconi Anemia BRCA (FA/BRCA)
deficient and corrected cell lines. FA/BRCA is a DSBR mechanism. The C1 graph shows the results of a cell survival
assay in increasing concentrations of MMAIII. Cell viability decreased in corrected and deficient cells lines in a
concentration dependent manner, with the greatest decrease clearly seen at subtoxic levels (red arrows). In C2, another
cell survival assay was conducted on corrected and deficient lines but in the presence of a cross-linking agent
mitomycin-C (MMC). In the presence of MMAIII, cell viability significantly dropped for corrected lines in a MMC
concentration dependent manner. The most significant drop was observed early on with the dose of MMC and there was
little difference between 0.05 and 0.1μM MMAIII [19]. The presence of MMAIII inhibited the ability of the FA/BRCA
cells to repair the DSB induced by MMC. The authors of this investigation stated a probably reason for this inhibition
was due to arsenic displacing zinc in the binding domains of PARP-1, leading to inhibited DNA repair [19]. The D1 plot
was provided by researchers showing AsIII-induced ROS/RNS generation causes zinc loss and inhibits the activity of
PARP-1 in immortalized human keratinocyte (HaCat) and normal human epidermal keratinocytes neonatal (HEKn) cells
[21]. In D1, the DNA binding ability of isolated and purified PARP-1 from untreated HaCat cells was analyzed utilizing
an electrophoretic mobility shift assay. The relative binding ability of PARP-1 decreased in a concentration dependent
fashion, with the greatest decrease at the lowest subtoxic AsIII levels [21]. The researchers of the E1 and E2 plots
conducted PARP-1 analysis from HEKn and normal human neonatal epidermal melanocytes (HEMn) and found both
cell types share a distinct response to iAs and UV light [20]. In E1, arsenite inhibited PARP-1 activity in both cell types
in a concentration dependent fashion in the presence of UV utilizing an HT colorimetric PARP/Apoptosis assay kit. The
greatest decrease in PARP-1 activity was observed at lower levels [20]. In E2, the zinc content in both cell types was
decreased in the presence of UV and increasing concentrations of arsenite, utilizing a zinc release assay [20]. There was
a significant decrease in zinc content in both cell lines at the lowest level doses, consistent with the majority of
experiments conducted on PARP-1 as well as assessing non-carcinogenic endpoints associated with iAs exposure.
Regulation, Remediation & Proposed Regulation
Due to the prevalence of AsV and AsIII species along with other hazardous contaminants in potable water sources,
MCLs are implemented by the United States Environmental Protection Agency (EPA) to protect public health. MCLs are
setup for contaminants based on toxicological studies and economic feasibility. The MCL for iAs is10 ppb [23].
The EPA has verified the performance of twelve technologies for remediating iAs and is currently exploring passive
treatment with a permeable reactive barrier (PRB) [24,25]. A recent study suggests that the use of nickel smelter slag as a
reactive medium for site remediation in a PRB is promising [26]. Studies are also being conducted on bioremediation
tools as a safe approach for iAs remediation in an effort to control disposal costs [27]. Also, a less expensive and
environment-friendly method has been studied as a possible pre-treatment step before large chemical treatment
decontamination, utilizing an agricultural waste maize powder [28].
On June 22, 2000, the EPA originally drafted the revised MCL for iAs to be 5 ppb before the final ruling was set to 10
ppb [23]. Since this last update to the MCL, strong data from epidemiological and animal studies have shown the
standard to be inadequate in protecting against long-term chronic exposures. The above figures show the greatest adverse
cellular effects from iAs exposure to occur at apparently subtoxic concentrations in all studies. Therefore, the proposed
standard, based on best available science and technology, is 5 ppb; this is the same standard USEPA wanted to pass over
a decade ago but, due to feasibility and best available science fourteen years ago, the standard could not be met.
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