Accepted Manuscript

Oxidative stress during development: Chemical-induced teratogenesis

Jason M. Hansen, Benjamin R. Jacob, Ted B. Piorczynski

PII: S2468-2020(17)30124-9
DOI: 10.1016/j.cotox.2017.11.003
Reference: COTOX 98

To appear in: Current Opinion in Toxicology

Received Date: 10 October 2017

Revised Date: 24 October 2017
Accepted Date: 3 November 2017

Please cite this article as: J.M. Hansen, B.R. Jacob, T.B. Piorczynski, Oxidative stress during
development: Chemical-induced teratogenesis, Current Opinion in Toxicology (2017), doi: 10.1016/
j.cotox.2017.11.003.

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 ACCEPTED MANUSCRIPT

Oxidative stress during development: Chemical-induced teratogenesis

Jason M. Hansen1*, Benjamin R. Jacob1 and Ted B. Piorczynski1

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Department of Physiology and Developmental Biology
College of Life Sciences
Brigham Young University

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Provo, Utah, USA
84602

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*Corresponding author
4005 Life Science Building
Department of Physiology and Developmental Biology

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College of Life Sciences
Brigham Young University
Provo, Utah, USA
84602

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Jason M. Hansen: jason_hansen@byu.edu
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Benjamin R. Jacob: ben.richard.jacob@gmail.com
Ted B. Piorczynski: ted.piorczynski@gmail.com
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Abstract

Oxidative stress has been shown to be an important contributor to birth defects,

including structural malformation and neurobehavioral deficits. At high levels,
oxidative stress promotes apoptosis but at lower levels that do not cause cell
death, redox-sensitive pathways can be disrupted. Chemicals that cause birth
defects usually provide a very distinct developmental outcome, dependent upon

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gestational periods of exposure and level of exposure, but these observations
also suggest that there are specific pathways involved in the manifestation of a
given defect. Here, we review overarching themes of oxidative stress and their

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effects during development and provide specific examples of some of the most
notorious human teratogens that produce oxidative stress as part of their
teratogenic mechanism. Moving forward, future studies should center on

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providing a more profound understanding of redox-sensitive elements during
development to develop potential preventative interventions.

Keywords: Glutathione, Birth Defects, Oxidative Stress, Redox, Signaling,

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Apoptosis
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Abbreviations:

ASK1: Apoptosis Signaling Kinase 1

Cys: Cysteine
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D3T: 3H-1,2-dithiole-3-thione
Eh: Redox Potential
FASD: Fetal Alcohol Spectrum Disorder
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Grx: Glutaredoxin
GSH: Glutathione
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GSSG: Glutathione Disulfide

H2O2: Hydrogen Peroxide
MAP: Mitogen-activated Protein
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NAC: N-Acetylcysteine
NF-κB: Nuclear Factor Kappa B
Nrf2: NF-E2p45-related factor 2
ROS: Reactive Oxygen Species
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-SOH: Sulfenic Acid

-SSG: S-Glutathionylation
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Trx1: Thioredoxin-1
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Highlights:

• Redox states control important cell functions (e.g. proliferation, differentiation,

etc.)
• Oxidative stress can disrupt developmental pathways and lead to teratogenesis
• Many known human teratogens are also known oxidants

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1. Introduction

Alarmingly, one in every 33 births present with a birth defect, ranging from
relatively minor, non-life-threatening effects to severe, lethal developmental
outcomes. Unfortunately, upwards of 70% of all birth defects occur through
unknown etiologies [1]. Chemicals that produce birth defects, known as
teratogens, contribute only a small amount to the total known causes of birth

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defects in humans, but this may be an underestimate of environmental influences
in poor developmental outcomes. Of the known human teratogens, many are
known to produce oxidative stress [1], suggesting an important mechanistic link

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between oxidative stress, redox signaling and development. Here, in this review,
we outline the role of oxidative stress in developmental toxicity and chemical-
induced embryopathy.

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2. Oxidative stress and redox signaling

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Oxidative stress has been classically defined as an imbalance between
oxidizing and reducing equivalents, where the former predominates [2]. In
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severely pro-oxidizing environments, cellular macromolecules are damaged
promoting apoptosis or necrosis [2]. With less toxic levels of oxidative stress,
redox states are simply shifted but can disrupt or delay normal cell function. In
some cases, these redox-sensitive pathways are linked to systems that support
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the restoration of functional cellular redox states, prior to the oxidation-induced

promotion of cell death (Figure 1). However, there are other redox-sensitive
pathways that are likely more closely tied to developmental pathways, where
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untimely shifts in cellular redox states could result in aberrant signaling of redox-
sensitive pathways, support untimely cellular function and ultimately promote
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dysmorphogenesis.
Development is a mixture of different cellular actions, such as proliferation
and differentiation, which occur in a well-coordinated manner, where disruption of
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these events can result in poor developmental outcomes. Proliferative,

differentiative and apoptotic activities are, in part, regulated by cellular redox
states [3] and as such, can be altered during periods of oxidative stress and
redox imbalance. Perhaps the most susceptible periods in the embryo are
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periods defined by transition from an important developmental event to another

(e.g. proliferation→differentiation) in developing target organs and systems
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(Figure 2). Chemical-induced oxidative stress can result in prolonged periods of

redox imbalance that alter developmental programming from regular schedule
events.

3. Redox regulation during development

The intracellular redox state has been shown to be an integral part of

many essential developmental processes. The influence that redox states have
on development is complex and far reaching, affecting a variety of cellular
systems. Because cellular processes affect the redox state of the cell, the redox
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regulation of these processes can involve feedback control [4]. Reactive oxygen
species (ROS) are understood to have beneficial as well as deleterious effects
on embryo development. However, aberrant production of ROS to toxic levels
can occur through a variety of mechanisms including changes to cellular
metabolism, alterations to mitochondrial kinetics and the bioactivation of
chemicals to more reactive, oxidizing metabolites (e.g. ethanol is metabolized to
reactive acetaldehyde via alcohol dehydrogenase). Loss of ROS regulation can

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be damaging during development and thus, a tightly controlled redox state is
optimal for normal embryogenesis [5]. While there are many cellular processes
that rely on redox-dependent mechanisms, we have highlighted only three in this

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review that are most relevant to developmental toxicity-related processes.

3.1 Cellular signaling

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Cellular signaling has been widely shown to incorporate ROS, like H2O2,
as a signaling intermediate and secondary messenger. For example, epidermal
growth factor signaling produces H2O2 to oxidize proteins in tyrosine kinase

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cascades and promote DNA synthesis and cellular proliferation [6]. Similar
results were found with platelet-derived growth factors when their tyrosine
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receptor signaling systems were blocked by the inhibition of ROS production,
indicating the use of redox shifts in cellular signaling [7]. Furthermore, H2O2 is a
necessary intermediate for glucose-induced insulin secretion [8]. During
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development, the relative value of oxygen in metabolism fluctuates as the

preferential source for cellular energy shifts from glycolysis to oxidative
phosphorylation. This change shifts the redox state of the cell when the embryo
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uses mitochondrial production of ROS during metabolic switching [9, 10].

Additionally, H2O2 has been shown to activate the transcription factor NF-
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E2p45-related factor 2 (Nrf2) during times of electrophilic and oxidative stress.

Nrf2 then signals the increased gene expression of antioxidant and phase II
detoxification enzymes [11]. The Nrf2-mediated response is an important
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element of the antioxidant defense system in developing embryos. In some

models, embryos show distinct, inherent changes to redox states during
development, where cellular redox states during periods of differentiation are
generally more oxidizing than those observed during proliferation [12]. These
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changes may be an influencing factor in how Nrf2 is regulated during

development after oxidant exposure [13]. There are a number of transcription
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factors, like Nrf2, that are controlled by redox states, many of which are involved
in developmental events, and thus, support redox regulation involvement in
development [14, 15]. The specific mechanisms for the redox-sensitive signaling
methods are currently under investigation, but may be directly or indirectly
tethered to intracellular redox states. While S-thiolation has been suggested as a
redox-control mechanism, S-glutathionylation (-SSG) is seen as a direct control
regulator of redox-dependent responses [16]. Indirectly, loss of reducing power
may promote the increased availability of ROS to cause protein oxidation
(development of sulfenic acids, -SOH). As such, through disulfide exchange
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mechanisms, both indirect and direct redox control of proteins can overlap [17].

3.2 Differentiation

Cellular differentiation relies on some of the cellular signaling mechanisms

previously described and subsequently is also influenced by the redox potential
of the cell. Human stem cell differentiation into adipocytes and osteocytes has

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been shown to be directly related to the glutathione (GSH) redox potential (Eh);
in both processes, the overall GSH Eh shifted to a more oxidizing state, albeit at
different rates during the differentiation period [18] and may highlight specific

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redox regulation into certain phenotypes. Although redox states were not the
primary measure, studies show that mitochondrial ROS seem to be involved in
CD4+ and CD8+ memory T cell differentiation [19]. Reactive oxygen species

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have also been implicated as a signal of adipocyte differentiation [20, 21] and
osteocyte differentiation [22]. Reducing ROS levels in hematopoietic stem cells
promotes a more reducing environment which correlated with a restriction in the
cell’s ability to differentiate [23]. These findings suggest a more nuanced role for

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shifts in intracellular redox potentials and indicate that, at different redox
potentials and at different stages of development, an embryo can be more or less
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prone to differentiate.

3.3 Apoptosis
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Redox state is also linked with the activation and stimulation of apoptosis
[24, 25]. Reduced thioredoxin, a protein oxidoreductase, can bind to and inhibit
apoptosis signaling kinase 1 (ASK1), a member of the MAP3 kinase family [26].
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However, upon oxidation, Trx1 becomes unbound and allows ASK1 to become
fully active promoting apoptosis [27]. As such, changes in cellular redox
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environments specific to Trx1 Eh can directly influence ASK1 activity and

stimulate related downstream apoptotic activities.

As GSH becomes oxidized to glutathione disulfide (GSSG), protein targets

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can become S-glutathionylated and as a post-translational modification, protein

function can be changed. In relation to apoptosis, studies have shown that in
Fas-mediated death pathways, Fas can be glutathionylated on a redox-sensitive
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cysteine residue (Cys294) further promoting its downstream pro-apoptotic

signaling [28]. Fas-SSG increase FasL binding and accentuated apoptosis.
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Removal or addition of GSH moieties to proteins can occur through the action of
glutaredoxins (Grx), but mostly de-glutathionylate proteins under normal
conditions. For de-glutathionylation reactions, Grxs will reduce proteins and
become glutathionylated itself (Grx-SSG). Overexpression of Grx prevented the
formation of Fas-SSG and reduced apoptosis. As such, the role of GSH Eh
requires further study to better understand the proposed mechanisms here,
especially in the context of development.

4. Disruption of Redox Control during development through chemical

exposures
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Alterations in redox status influence multiple cellular processes that may

negatively affect embryonic development. Consequently, embryonic cells are
sensitive to the direct action of certain drugs or to the environmental changes
produced by drugs that induce oxidative stress [29]. While multiple teratogens
have been shown to increase ROS production and induce oxidative stress, we
will focus on only a few of the most prevalent ones for this review.

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4.1 Thalidomide

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In the 1950s, this non-addictive sedative was widely prescribed to
pregnant women to ameliorate nausea associated with morning sickness. Shortly
thereafter, thalidomide exposure during the first trimester of pregnancy was

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shown to be responsible for a number of deformities in newborns, including
phocomelia and amelia [30, 31]. Recently, interest in thalidomide has resurfaced
as it has received FDA approval as an effective therapy for multiple myeloma and
erythema nodosum leprosum [32]. Multiple sources have shown that thalidomide

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increases ROS production, induces oxidative stress and disrupts the function of
the redox-sensitive transcription factor, Nuclear Factor Kappa B (NF-κB), an
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important mediator of limb bud outgrowth, and appear to contribute to limb
reduction defects [33-36]. Pretreatment with various antioxidants reduces the
associated thalidomide-induced terata in animal models and restores redox
states to preserve normal cellular signaling and function [37, 38], further
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suggesting the role of oxidative stress, ROS generation and redox imbalance in
thalidomide teratogenesis.
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4.2 Phenytoin
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Phenytoin is a widely used anticonvulsant drug that induces oxidative

stress [39] and is associated with an increased risk for isolated cleft palate in
children following maternal exposure during the first trimester of pregnancy [40].
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In addition, phenytoin results in oxidative DNA damage and dysmorphogenesis,

which can be reduced by the antioxidant superoxide dismutase [41]. Phenytoin-
induced defects in animal models may be reduced through pretreatment with N-
acetylcysteine (NAC), a precursor to GSH synthesis. Conversely, pretreatment
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with antioxidant depleting agents increased the occurrence of defects [39].

Curcumin, a pigment from plants known to have antioxidant properties, is
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effective in preventing phenytoin-induced cognitive impairment and oxidative

stress in rats [42, 43]. In other studies, GSH levels in fetal organs were
significantly depleted when phenytoin was given from gestational day 7-18, yet
interestingly, antioxidant supplementation (vitamin E), in this case, failed to
rescue changes to GSH concentrations [44].

4.3 Ethanol
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Ethanol consumption during pregnancy can result in a wide range of

adverse effects in the developing fetus. Fetal Alcohol Spectrum Disorder (FASD)
comprises the vast range of conditions that can occur when alcohol is consumed
during various periods of pregnancy. Fetal Alcohol Syndrome (FAS), the most
severe condition, is characterized by facial abnormalities, growth retardation, and
central nervous system impairment [45]. Evidence from in vitro and in vivo
models of prenatal ethanol exposure suggest that oxidative stress contributes to

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deficits seen in FASD, and that antioxidants are potential candidates for the
treatment of alcohol-induced developmental disorders [46]. In In vitro culture,
embryos demonstrated a decrease in GSH concentrations with ethanol exposure

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and similar GSH depletion was observed with in utero exposure [47]. In the
same study, embryonic cysteine was also significantly decreased but to an even
greater extent than GSH with both in vitro and in utero exposures. Ethanol

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caused an increase in H2O2 and superoxide production in cultured fetal
hepatocytes, as well as an increase in malondialdehyde, a marker of lipid
oxidative damage [48, 49]. The Nrf2-inducer, D-L-sulforaphane, protects neural
crest cells, an ethanol-sensitive cell population implicated in FASD, against

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ethanol-induced oxidative stress and apoptosis [50]. Furthermore, in utero,
administration of the Nrf2 inducer, 3H-1,2-dithiole-3-thione (D3T), improved
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antioxidant enzyme expression and activities and prevented cranial apoptosis in
the embryonic neural folds [51].

4.4 Mercury
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Mercury is a naturally occurring element found in the earth’s crust that has
been released into the biosphere as a consequence of natural processes
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including volcanic activity, fires and erosion. Recently, anthropogenic sources

have greatly contributed to the environmental distribution of mercury via coal
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mining and chemical syntheses. While various types of mercury occur in nature,
perhaps the most detrimental, both developmentally and otherwise, is
methylmercury, the most abundant organic mercury. Methylmercury has been
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shown to bioaccumulate in organisms causing severe health issues including

irreversible brain damage and even death [1]. Fetal methylmercury poisoning has
been well documented in Japan, where mothers consumed fish contaminated by
methylmercury resulting in numerous children being born with neurobehavioral
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deficits [52]. In mice, various in utero effects were observed with methylmercury
exposure, including cleft palate, exencephaly, missing limbs and facial
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deformities [53]. Microglial cells exposed to methylmercury showed a

concentration- and time-dependent increase in ROS generation and a
concomitant decrease in the ratio of GSH and GSSG [54]. In a mouse model,
administration of NAC prevented methylmercury-induced malformations [55]. In
mouse fetuses, methylmercury caused a significant increase in GSSG and
decrease in GSH, supporting an environmental redox shift correlation with its
developmental toxicity [56]. Interestingly, mercury exposure did not increase
GSH synthesis pathways in the fetus as the GSH synthesis enzyme activity was
unchanged, but increases in activity were observed in the visceral yolk sac,
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which may suggest poor oxidant pathway responses in the embryo/fetus during
these stages of development.

5. Conclusion

During development, cells are in flux, as demonstrated by specified

periods of proliferation, differentiation and apoptosis. Oxidant exposure can

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increase ROS levels resulting in variable effects. Dependent upon the degree of
ROS generation, cellular macromolecules can be severely or irreversibly
damaged to the point where cells will undergo apoptosis, whereas under lower

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ROS conditions, redox states can be altered to modify redox-sensitive signaling
pathways (Figure 3). With the latter, there are pathways that favor the correction
and restoration of redox states to reverse redox imbalanced cellular

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environments. However, during periods of redox imbalance, developmental
stage specific redox-sensitive pathways can still be perturbed, resulting in failed
developmental programming and poor developmental outcomes such as
structural malformations and/or neurobehavioral deficits. Clearly, moving forward

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to better understand environmental influences, both physical and chemical, that
act to disrupt timely developmental signaling through dysregulation of redox
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states is an area of great human health interest and should be further studied to
develop intervention and prevention strategies.
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Figure legend:

Figure 1. Comparison of the activation of redox-sensitive pathways (green line)

with pro-cell death (red line) signaling. Under normal, usually pro-reducing,
intracellular redox states, compensatory redox signaling and cell death is low. As
redox states shift to a more oxidizing environment, as would occur with chemical-
induced oxidative stress, redox-sensitive elements and pathways would be

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activated earlier in the redox spectrum and at more moderate redox shifts. As
redox states progress to more pro-oxidizing potentials, various redox-sensitive
signaling pathways could be deactivated dependent on their redox sensitivity. As

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these corrective pathways are either shut off or deactivated, apoptotic signaling
will predominate to promote cell death.

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Figure 2. Redox switches involved in cellular function. Under highly reducing
conditions, proliferation-related pathways are switched on (green line), but as
redox states transition to a more oxidizing condition, pro-proliferation switches
are deactivated and pro-differentiation switches (blue line) are activated.

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Excessive shifts to extremely pro-oxidizing redox states switch off pro-
differentiation pathways and promote apoptosis (red line) and necrosis (black
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line). During development, transitional periods (outlined in yellow) may be the
most susceptible periods to chemical-induced redox signaling disruption.
Untimely switching may prevent normal developmental processes and cause
poor developmental outcomes (structural malformations, neurobehavioral
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deficits, etc.).

Figure 3. Overview of oxidative stress in developing tissues. Following chemical

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exposure, changes to cellular metabolism, mitochondrial kinetics or the

bioactivation of xenobiotics into reactive metabolites can promote an increase in
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ROS production increases and shift intracellular redox states. Excessively high
levels of ROS generation and/or highly oxidized redox states can promote
apoptosis (black arrows), but under more moderate conditions, shifts in redox
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state can promote disruption to redox-sensitive cell signaling (red arrows),

including important developmental pathways. Outcomes can include structural
malformations and neurobehavioral deficits. However, redox shifts can also
activate compensatory pathways that are designed to restore redox imbalance
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and increase ROS detoxification (green arrows). Rapid and timely restoration of
redox states may serve as a means to prevent prolonged periods of signal
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disruption and support normal developmental patterning.

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