TOXICOLOGICAL SCIENCES, 143(2), 2015, 242–255

doi: 10.1093/toxsci/kfu233
REVIEW

Molecular Mechanisms of Acrolein Toxicity: Relevance

to Human Disease

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Akshata Moghe*, Smita Ghare†, Bryan Lamoreau†, Mohammad Mohammad†,
Shirish Barve*,†, Craig McClain*,†,‡, and Swati Joshi-Barve*,†,1
*Department of Pharmacology and Toxicology, †Department of Medicine and ‡Robley Rex VAMC, Louisville,
Kentucky 40202
1
To whom correspondence should be addressed at Department of Medicine, University of Louisville, 505 S. Hancock Street, Room 505 CTRB, Louisville,
Kentucky 40202. Fax: 502-852-8927. E-mail: s0josh01@louisville.edu.

ABSTRACT
Acrolein, a highly reactive unsaturated aldehyde, is a ubiquitous environmental pollutant and its potential as a serious
environmental health threat is beginning to be recognized. Humans are exposed to acrolein per oral (food and water),
respiratory (cigarette smoke, automobile exhaust, and biocide use) and dermal routes, in addition to endogenous
generation (metabolism and lipid peroxidation). Acrolein has been suggested to play a role in several disease states
including spinal cord injury, multiple sclerosis, Alzheimer’s disease, cardiovascular disease, diabetes mellitus, and neuro-,
hepato-, and nephro-toxicity. On the cellular level, acrolein exposure has diverse toxic effects, including DNA and protein
adduction, oxidative stress, mitochondrial disruption, membrane damage, endoplasmic reticulum stress, and immune
dysfunction. This review addresses our current understanding of each pathogenic mechanism of acrolein toxicity, with
emphasis on the known and anticipated contribution to clinical disease, and potential therapies.

Key words: antioxidants; apoptosis; DNA adducts; inflammation; exposure; environmental; oxidative injury

Acrolein (CAS No 107-02-8) is listed by the Department of
Homeland Security (DHS), Agency for Toxic Substances and
Disease Registry (ATSDR), and Environmental Protection
Agency (EPA) as a high priority toxic chemical (Toxicological
Profile For Acrolein, 2007), with recommendations on acrolein
exposure levels in the environment, food, and water. Acrolein is
an industrial chemical and a biocide, and over 500 million
pounds of acrolein are produced annually in the United States
(Toxicological Profile For Acrolein, 2007). The past decade has
witnessed a dramatic increase in research regarding acrolein,
and this review attempts to assimilate this growing literature
with a focus on the molecular mechanisms of acrolein toxicity
pertaining to human disease. Although some early literature
has been cited, this review is focused on more recent literature.
The theme of this review is that there are multiple mechanisms
involved in acrolein toxicity. In our extensive appraisal of the
current literature, our goal was not only to characterize these
various mechanisms, but also to identify an overarching path-
way through which acrolein toxicity may be initiated and
propagated. Our analysis suggests that such a common unifying
pathway might not exist or is not currently discernable, placing
even more onus on the need to further investigate acrolein tox-
icity in various disease conditions. This comprehensive review
starts with succinct introductory comments on the generation,
sources, and metabolism of acrolein, followed by a detailed dis-
cussion on the various mechanisms of acrolein toxicity, which
include protein adduction, oxidative stress, mitochondrial dys-
function, DNA adduction, endoplasmic reticulum (ER) stress, in-
flammation and immune alterations, structural and membrane
effects, and dysregulated signal transduction.
ACROLEIN SOURCES AND METABOLISM
Sources. Acrolein is a common dietary and environmental pollu-
tant and is also generated by cellular metabolism (Figure 1). It is
an a,b-unsaturated aldehyde released during the combustion of
petroleum fuels, biodiesel, plastic, paper and wood, and is a
major component of tobacco smoke; such exposures are directly

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242

Endogenous
Lipid Peroxidation
Anti-Cancer Drugs Alcoholic beverages
Polyamines
Threonine
Protein and
HPMA
DNA Adducts
METABOLITES
FIG. 1. The sources and metabolic fate of acrolein. GSH: glutathione; CEMA: 2-carboxyethylmercapturic acid; HPMA: 3-hydroxypropylmercapturic acid; OPMA: S-(3-oxo-
propyl)-N-acetylcysteine.
relevant in dermal and respiratory toxicity. Firefighters, acrolein
industry workers, persons inhabiting densely polluted cities,
and cigarette and hookah smokers (Kassem et al., 2014) face sub-
stantial exposures (reviewed in Cahill (2014); Stevens and Maier
(2008)). Acrolein has been identified as a health hazard com-
monly found at significant levels in most homes and schools in
the United States (Logue et al., 2012). Acrolein can be generated
from overheated vegetable and animal fats; hence, cooked,
fried, and charred foods are notable sources of acrolein, as are
beer, wine, rum, and breads; the best current estimate for
Tolerable Daily Intake (TDI) for acrolein is 7.5 mg/kg body weight
(reviewed in Abraham et al. (2011)). Moreover, acrolein exposure
from consumption of foods may be considerably underesti-
mated (Watzek et al., 2012). Exposure to reactive unsaturated
aldehydes like acrolein by dietary consumption is estimated at
nearly 5 mg/kg of food (Wang et al., 2008). The gastrointestinal
(GI) system is likely affected the most by ingested acrolein.
Notably, acrolein can also be generated endogenously during
cellular metabolism by (1) degradation of threonine by neutro-
phil-derived myeloperoxidase; (2) amine oxidase-mediated
catabolism of polyamines such as spermine and spermidine; (3)
metabolism of cancer drugs such as cyclophosphamide; and (4)
lipid peroxidation (LPO) of polyunsaturated fatty acids (PUFAs)
(Stevens and Maier, 2008; Uchida et al., 1998a,b). The levels of
acrolein formed by metabolism are difficult to quantify, and
may reach very high levels in certain cellular microenviron-
ments; hence, a determination of adverse effects is challenging.
Importantly, both acute and chronic acrolein exposures are
described to have significant adverse effects (Centers for
Disease and Prevention, 2013).
Metabolism. Acrolein is highly water soluble and can rapidly enter
bodily tissues. It is known to rapidly form conjugates with cellular
glutathione (GSH), cysteine, N-acetylcysteine (NAC), and/or thio-
redoxin. Acrolein-GSH conjugation can occur without a catalyst
MOGHE ET AL. | 243
SOURCES
Dietary Exogenous
Fried food Automobile Exhaust
Cigarette Smoke
Charred Meat Industrial Waste
Forest Fires
ACROLEIN
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GSH-Acrolein
Acrylic acids
OPMA CEMA
Glyceraldehyde
Malonic acid
or may be mediated by glutathione-S-transferases; these acro-
lein-GSH conjugates may be metabolized by enzymatic cleavage
of c-glutamic acid and glycine residues. Further metabolism via
aldehyde dehydrogenase results in 2-carboxyethylmercapturic
acid (CEMA), or catalyzed by aldo-keto reductase results in S-(3-
hydroxypropyl)-N-acetylcysteine (3-HPMA), the main urinary
metabolite of acrolein. Acrylic acid and glycidaldehyde are alter-
nate acrolein by-products, should oxidation and epoxidation pre-
cede its conjugation with GSH; glycidaldehyde may represent a
human health risk as it has carcinogenic properties in mice and
rats when applied dermally. Acrolein may be also converted to
malonic acid by aldehyde dehydrogenase. Finally, acrolein may
form irreversible adducts with cellular nucleophiles, a metabolic
reaction that may be the basis of several of its toxic effects
(Figure 1) (reviewed in Stevens and Maier (2008)).
MECHANISMS OF TOXICITY
An interesting feature of acrolein is the diversity with which it
exerts its toxic effects in different tissues and organs. Both dis-
ease- and organ-specific, as well as shared mechanisms have
distinct value in the search for therapeutic targets; the former
enables specific, targeted therapy, whereas the latter facilitates
the broad management of acrolein toxicity. In the following sec-
tions, the various direct mechanisms of acrolein toxicity such
as protein and DNA adduction, and indirect mechanisms
including induction of oxidative, mitochondrial, and ER stress,
are critically discussed with possible connection to affected
organs and pathological conditions. Wherever known or tested,
the effectiveness of acrolein scavengers and other compounds
in mitigating acrolein toxicity has also been described (Table 1).
Protein Adduction
Being a strong electrophile, acrolein shows high reactivity with
cellular nucleophiles such as proteins, DNA and RNA. Acrolein
 244 | TOXICOLOGICAL SCIENCES, 2015, Vol. 143, No. 2

TABLE 1. Acrolein Toxicity and Protective Agents

Mechanisms of Acrolein Toxicity Protective Agents

Protein adduction Hydralazine

Oxidative stress
Mitochondrial dysfunction
Inflammation and Immune Alterations
ER stress
Structural and membrane effects
Deregulated signal transduction
NAC, Hydralazine, MESNA, Lipoic acid, Hydroxytyrosol, Vitamin E, Diallyl disulfide, Rapamycin,
L-Cysteine, Penicillamine, Aminoguanidine, Hydroxylamine, Sodium Borohydride, Sodium
Bisulfite, Ascorbic acid, Trolox
NAC, Polyethylene glycol-catalase, SOD mimetic-MnTBAP, Pifithrin- p53 inhibitor
NAC, MESNA, U0126- AKT inhibitor, FR180204 - ERK1/2 inhibitor, Tetramethylthiourea
NAC, Caspase inhibitor, Phenylbutyric acid, JNK inhibitor
NAC, Hydralazine
NAC, Rottlerin- PKC-d inhibitor, JNK inhibitor, Simvastatin, Rosiglitazone, Sildenafil, NBHA
(N-benzylhydroxylamine), SIS3- SMAD3 inhibitor

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readily targets the sulfhydryl group of cysteine, the imidazole
group of histidine and the amino group of lysine, to form mainly
Michael addition-type adducts or Schiff base cross-links
(reviewed by Pizzimenti et al. (2013); Zarkovic et al. (2013)). These
susceptible amino acid residues have major physiological
importance and are involved in cellular processes including
enzyme catalysis, redox signaling, reactive oxygen species
(ROS) sensing, and cellular buffering; hence, adduction by acro-
lein may lead to significant alterations in protein function. Also,
acrolein can interfere with its own metabolism by inhibiting
two enzymes that metabolize acrolein-GSH conjugates,
namely, alcohol dehydrogenase and aldehyde dehydrogenase.
Furthermore, acrolein may augment the effects of other known
targets via irreversible adduction and inactivation of arylamine
N-acetyltransferases, a major family of xenobiotic-metabolizing
enzymes involved in metabolizing many drugs and pollutants
(Bui et al., 2013).
Acrolein protein adducts are assessed primarily by antibody-
based techniques, high-performance liquid chromatography
(HPLC), and mass spectrometry, and such adducts have been
demonstrated in vitro and in vivo in several cell types and tissues
(Aldini et al., 2011). Alterations in protein function caused by
acrolein adduction have been demonstrated in several studies,
albeit primarily in vitro. In 1998, Uchida et al. were the first to
show that an acrolein-lysine adduct (FDP-Lys) was involved in
the oxidation of human low-density lipoprotein (LDL); they also
demonstrated the presence of specific antibodies against this
adduct in vivo (Uchida et al., 1998b). Prabhu’s group showed that
direct exposure to acrolein (as low as 0.01 mM) induced myofila-
ment dysfunction in vitro and in vivo (1 mg/kg/day intravenous)
in mice (Ismahil et al., 2011), with accumulation of acrolein-pro-
tein adducts and protein carbonyls within the myocardium.
Burcham et al. showed in mouse hepatocytes that exposure to
allyl alcohol and its metabolite acrolein led to protein carbony-
lation, which correlated with hepatocyte death. They later
showed that the acrolein scavenger hydralazine effectively
trapped acrolein adducts in vivo to reduce allyl alcohol-
mediated hepatotoxicity in mice, and that mobilization of the
chaperone protein HSP90 was protective against acrolein-
induced carbonylation (Burcham et al., 2012). Acrolein was
suggested to play a role in liver injury in a murine model of
early alcoholic liver disease and two novel in vivo protein targets
for acrolein were identified: neutral alpha-glucosidase AB and
nesprin-1 (Galligan et al., 2012). Treatment of recombinant rat
apoE with a 10-fold molar excess of acrolein resulted in an
impairment in cholesterol efflux abilities, and a significant
decrease in lipid-binding and LDLr- and heparin-binding capa-
bilities, and overall stability of the protein (Tran et al., 2014).
Recently, it was demonstrated that salivary lactate
dehydrogenase (LDH) activity was reduced by cigarette smoke
(34%), and more so by 10 mM acrolein (61%) via introducing a car-
bonyl group into the protein and causing dysfunction; this
established acrolein as the main ingredient in cigarette smoke
responsible for salivary LDH activity diminution (Avezov et al.,
2014a).
A pathological role for acrolein in disease development/
progression has been proposed based on the detection of higher
levels of acrolein adducts in the sera of patients with various
diseases, including diabetic nephropathy; renal disease;
Sjogren’s syndrome autoimmune disorder (Brooks, 2013); cere-
bral stoke/infarction (Igarashi and Kashiwagi, 2011); colon carci-
nogenesis; and diabetes (reviewed by Aldini et al. (2011)).
A recent study using sera from patients with diabetic nephrop-
athy described the utility of “aldehyde capture”, a procedure
used to calculate the protein carbonyl stress derived solely from
LPO, suggesting future promise in the monitoring of renal dis-
ease and treatment (Medina-Navarro et al., 2011). The potential
utility of plasma levels of protein-conjugated acrolein (along
with amyloid b40/42 (Ab40/42)) to detect and differentiate
between mild cognitive impairment and Alzheimer’s disease
was recently demonstrated (Mizoi et al., 2014). Thus, substantial
evidence suggests that acrolein toxicity in major organ systems
involves the common mechanism of formation of adducts with
proteins that play critical functional roles in cellular processes.
However, the question of causality versus association still
remains, and the direct/indirect mechanisms by which the
acrolein adducts may lead to injury remain to be determined.
Oxidative Stress
Aerobic metabolism and pathogen defense mechanisms pro-
duce ROS which can damage lipids, proteins, carbohydrates,
and nucleic acids. To counter oxidative damage, the human
body is equipped with antioxidant compounds and antioxidant
enzymes. However, extensive ROS production can overwhelm
these defense mechanisms, and results in oxidative stress
(reviewed by Birben et al. (2012)). Acrolein is considered both a
product and an initiator of LPO (Adams and Klaidman, 1993;
Uchida et al., 1998a) and is thus a perpetrator of oxidative stress.
Both in vitro and in vivo studies have shown that acrolein can
itself cause oxidative damage, leading to membrane disruption,
DNA and mitochondrial damage and can exacerbate apoptosis.
Acrolein exposure (0.1–5 mM) in the human retinal epithelial
cells (ARPE19) significantly increased oxidant levels by decreas-
ing GSH, anti-oxidant enzymes (SOD and GSH-peroxidase), and
total and nuclear levels of the antioxidant regulator-Nrf2, coin-
cident with reduced mitochondrial function, suggesting a role
for acrolein in retinal damage and age-related macular degener-
ation. Notably, lipoic acid was protective (Jia et al., 2007), as was

hydroxytyrosol (antioxidant polyphenol in olives) via induction
of phase II detoxifying enzymes and stimulation of mitochon-
drial biogenesis (Zhu et al., 2010), and a-tocopherol (Vitamin E)
by upregulating Nrf2 (Feng et al., 2010).
With the abundance of PUFAs available for LPO and the lack
of enzymes that metabolize reactive aldehydes, neurons and
the central nervous system are particularly vulnerable to oxida-
tive stress and acrolein toxicity (Hamann and Shi, 2009).
Acrolein-induced neuronal damage was demonstrated in pigs
and rats, suggesting that it may be a potential risk factor for spi-
nal cord injury (SCI) (Due et al., 2014; Shi et al., 2011; Zheng et al.,
2013). The neuroprotective role of hydralazine was shown in the
attenuation of acrolein-mediated damage in SCI (Park et al.,
2014). Plasma levels of protein-conjugated acrolein (along with
acrolein producing enzymes—spermine oxidase and acetylpoly-
amine oxidase) were shown to be good biomarkers for human
stroke. Interestingly, an inverse correlation was seen between
stroke and 3-HPMA the urinary GSH-metabolite of acrolein; the
authors proposed that the decrease in 3-HPMA reflects reduced
GSH caused by increased acrolein generation at the region of
brain infarct (Yoshida et al., 2012).
Acrolein-induced oxidative stress is associated with severe
toxicity in the renal system due to the use of anticancer agents
such as cyclophosphamide and ifosfamide that get
metabolized to acrolein; the acrolein toxicity is prevented by
Mesna (2-mercaptoethane sulfonate) which binds with and
clears acrolein. A major contributor to acrolein toxicity in such
cases appears to be the activation of intracellular ROS and nitric
oxide production, either directly or via NF-jB and AP-1 (Korkmaz
et al., 2007). A recent report showed that pretreatment with diallyl
disulfide protected against cyclophosphamide-induced oxidative
damage, histopathological lesions, and apoptosis; notably, the
accumulation of acrolein-adducts in the bladder was reduced
showing that the protection was by detoxifying acrolein (Kim
et al., 2014). A recent review identifies acrolein-lysine adducts as a
promising urinary biomarker of oxidative stress and oxidative
damage (Il’yasova et al., 2012). Acrolein-induced oxidative dam-
age is shown to have an indirect impact on cardiovascular health
via apoptosis of adult mice cardiomyocytes in vitro (Wang et al.,
2011); and via myocardial oxidative stress, increased apoptosis,
and myocyte hypertrophy and dilated cardiomyopathy in vivo
after long-term (48-day) oral exposure of mice to 1 mg/kg acrolein
(Ismahil et al., 2011). Also, acrolein-induced oxidation of apolipo-
protein A-I (apoA-I, the major high density lipoprotein) impaired
cellular cholesterol efflux (Shao, 2012). The exposure of oral
tissues to environmental hazards is immense, especially in
smokers, and acrolein was shown to reduce GSH, and increase
oxidative stress and protein carbonylation in oral keratinocytes
in vitro (Avezov et al., 2014b). Studies in chronic obstructive
pulmonary disease (COPD) have shown that cigarette smoke
depletes total GSH levels in bronchial cells (van der Toorn et al.,
2007) and human gingival fibroblasts (Colombo et al., 2012); in
both cell types, mass spectrometric analyses demonstrated that
the GSH was depleted by binding to acrolein. Rapamycin was
demonstrated to inhibit acrolein-induced apoptosis in male germ
cells by alleviating acrolein-induced ROS, and reducing ROS-
driven mitochondrial dysfunction and apoptosis by increasing
ratio of Bcl2/Bax and protecting mitochondrial membranes (He
et al., 2014). Our work has shown that acrolein-induced oxidative
stress was accompanied by mitochondrial dysfunction and ER
stress in liver cells; acrolein toxicity was attenuated by the anti-
oxidant and GSH-prodrug NAC (Mohammad et al., 2012). A recent
article ranked the overall effectiveness of known protective
agents in suppressing acrolein-induced ROS formation, protein
MOGHE ET AL. | 245
damage, and toxicity in cell and cell-free models; starting
with the most effective: reducing agents > thiol-containing
compounds > carbonyl scavengers/amines > antioxidants/ROS
scavengers (MacAllister et al., 2013).
Like many other inducers of oxidative stress, acrolein is
known to activate cellular antioxidant systems, most likely
as an adaptive response. These effects are seen primarily at
low sublethal acrolein concentrations, and may be due to
“hormesis” described for other toxins, wherein hormesis-stimu-
lating compounds initiate an adaptive response that confers
protection against harmful doses of the same or similar agents.
One such example is the acrolein-induced activation of
Nrf2 in vitro in endothelial cells leading to induction of heme-
oxygenase-1, the enzyme that plays a major role in the cellular
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antioxidative response (Wu et al., 2006); and in vivo in the lungs
of mice exposed to 5 ppm of acrolein vapor for 6 h/day for 14–17
days (Zhang and Forman, 2008). The effects of acrolein on two
major redox regulators (thioredoxin and thioredoxin reductase)
have been recently discussed (Myers et al., 2011). In the event
that induction of cellular antioxidant systems fails or is subpar,
acrolein may induce pathological oxidative mechanisms that
contribute to disease development.
Mitochondrial Dysfunction
Mitochondria play a dynamic role in human cells, controlling
oxidative phosphorylation to produce ATP, aiding in fatty acid
breakdown and steroid synthesis, and playing an integral role
in calcium signaling and apoptosis. Acrolein has been impli-
cated as a mitochondrial toxin affecting several of these func-
tions, most notably apoptosis, and whether apoptosis or
necrosis ensues after acrolein exposure appears to be related to
dose and cell type. The majority of these studies are in vitro, and
the lack of much-needed in vivo data raises questions about
their relevance; nonetheless, some key studies are discussed
below.
Although the mitochondrial death pathway leading to apop-
tosis typically involves the activation of caspases, acrolein-
induced apoptosis was shown to be both caspase-dependent in
human neuroblastoma cells (Dong et al., 2013) and in A549 lung
cells (Roy et al., 2010), and caspase-independent in CHO cells
(Tanel and Averill-Bates, 2005). On the other hand, acrolein may
inhibit caspases and ultimately inhibit apoptosis in some cells,
such as murine proB lymphocytes (Kern and Kehrer, 2002) and
in B lymphoblastoid SKW6.4 cells via direct alkylation of cas-
pase active sites (Hristova et al., 2007).
Tanel et al. showed that NAC prevented apoptotic events in
CHO cells exposed to low doses of acrolein (4 fmol/cell, 30 min),
including Bad and Bcl-2 translocations, depolarization of the
mitochondrial membrane potential, procaspase-9 processing,
caspase-9, 7, 8 activation, and poly (ADP-ribose) polymerase
cleavage (Tanel and Averill-Bates, 2007b); however, apoptosis
was not prevented by the Fas receptor antagonist KP7-6 (Tanel
and Averill-Bates, 2007a). Roy demonstrated in A549 human
lung cells that low-dose acute acrolein exposures (3–27 mM,
30–60 min) could be overcome by an adaptive response involv-
ing activation of antiapoptosis survival factors AKT and cIAP1/2,
despite triggering of early apoptotic changes in Bax and cyto-
chrome c release; the survival proteins did not protect against
higher acrolein exposures (10–50 mM) induced apoptosis (Roy
et al., 2009). Most adverse changes were inhibited by catalase,
the antioxidant polyethylene glycol-catalase, and the SOD mim-
etic-MnTBAP, reiterating the initiating role of acrolein-induced
oxidative stress. Apoptosis in A549 lung cells was also reversible
with pifithrin-a, an inhibitor of p53 (Roy et al., 2010). Acrolein is
 246 | TOXICOLOGICAL SCIENCES, 2015, Vol. 143, No. 2
known to induce mitochondrial dysfunction leading to neuronal
death in HT22 mouse hippocampal cells, and lithium was
shown to protect against acrolein-induced neurotoxicity by
enhancement of GSH, and attenuation of ROS and mitochon-
drial dysfunction caused by acrolein (Huang et al., 2014).
Acrolein also impacts the ability of the mitochondria to take
part in cellular respiration. In rat liver mitochondria acrolein
dose-dependently inhibited select components of the respira-
tory machinery, including complexes I and II, pyruvate dehy-
drogenase, and a-ketoglutarate dehydrogenase, over a broad
range of acrolein exposures from 10 mM (relevant to human
exposures) to 1000 mM (highly unlikely) (Sun et al., 2006).
Mitochondrial dysfunction due to inhibition of key mitochon-
drial enzymes (pyruvate dehydrogenase and a-ketoglutarate
dehydrogenase) has been observed in Alzheimer’s disease brain
tissue; and lipoic acid, a cofactor for these enzymes, can be
covalently bound and sequestered by acrolein, thereby decreas-
ing enzyme activity. In keeping with this, supplementation with
lipoic acid was shown to decrease oxidative stress and mito-
chondrial enzyme loss, partly by scavenging of acrolein
(Maczurek et al., 2008). The presence of increased protein-
carbonyl adducts in this study emphasizes that particular mito-
chondrial enzymes may be more susceptible to covalent
modification, thereby acting as a wrench in the cogwheels of
electron transport.
DNA Adduction
DNA damage can be induced by exposure to chemicals, ROS and
reactive metabolites such as acrolein; DNA damage has been
associated with mutations and carcinogenesis. Acrolein can
produce interchain cross-links of double-stranded DNA as well
as DNA-protein cross-links. Acrolein readily reacts with deoxy-
guanosine (dG) producing two exocyclic DNA adducts, a- and
c-hydroxy-1, N2-propano-20 -deoxyguanosine (a-HOPdG and
c-HOPdG), which are mutagenic to varying degrees (Tang et al.,
2011). Additionally, acrolein adducts of 20 -deoxyadenosine
(Pawlowicz et al., 2006a,b) and 20 -deoxycytidine DNA bases and
thymidine (Pawlowicz and Kronberg, 2008) have been reported.
Acrolein-DNA adducts have been characterized in vitro, and
have also been detected in vivo in several different animal and
human tissues and cells (Tang et al., 2011; Voulgaridou et al.,
2011).
Acrolein adduction of DNA, if not repaired efficiently, has
the potential to cause critical gene mutations, suggesting that
acrolein may be mutagenic and may contribute to the process
of carcinogenesis. Mutagenicity studies of acrolein have yielded
inconsistent results, and both cell type and sequence context
appear to play a role (reviewed in Liu et al. (2010)). A lack of
mutagenicity by acrolein was demonstrated in various shuttle
vector-based systems (Kim et al., 2007); in contrast, Demir’s
group demonstrated dose-dependent in vitro mutagenic effects
of acrolein (10 and 25 mM) using the mouse lymphoma and the
Drosophila wing spot mutagenicity assay (Demir et al., 2013).
Furthermore, CpG methylation, at CpG-rich hot spots that are
prone to acrolein-DNA adduction, increased the acrolein-
induced G-to-T and G-to-A mutation frequency to levels found
in the CpG sites in the p53 gene, a known mutational hot spot in
tobacco smoke-associated lung cancer (Wang et al., 2013). In the
mitochondria, the natural absence of nucleotide excision repair
enzymes permits accumulation of acrolein adducts in mito-
chondrial DNA, leading to transversion mutations which are
thought to contribute to neurodegenerative disorders
(Kasiviswanathan et al., 2013). Interestingly, acrolein not only
induced DNA damage but also inhibited DNA repair by
inhibition of repair enzymes, likely by acrolein-adduction lead-
ing to increased proteasome-dependent degradation (Wang
et al., 2012) or slower repair activity (Choudhury et al., 2013).
Additionally, acrolein inhibited DNA methylase by 30%–50%
(Cox et al., 1988), and inhibited histone acetylation and compro-
mised chromatin assembly suggesting that acrolein exposure
may also cause epigenetic alterations in gene expression (Chen
et al., 2013). However, in vivo evidence of mutagenicity has not
been demonstrated thus far.
Acrolein-induced DNA damage and inhibition of DNA repair
are likely to promote cancer development; however, the carci-
nogenicity of acrolein is controversial (reviewed in Abraham
et al. (2011)). Intraperitoneal administration of acrolein (75 and
150 nmol per animal) increased DNA adducts but not tumor
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incidences in male or female B6C3F1 neonatal mice, suggesting
that DNA adduction may not parallel cancer development (Von
Tungeln et al., 2002). In contrast, a case has been made for the
carcinogenicity of acrolein based on higher acrolein DNA
adducts levels in lung (Feng et al., 2006), liver (Kawai et al., 2003),
and bladder cancers (Lee et al., 2014). In an early study, acrolein
was postulated to promote urinary bladder cancer since intra-
peritoneal and intravesicular administration of acrolein demon-
strated abnormal cell proliferation and hyperplasia of the
bladder epithelium and the urothelium (Cohen et al., 1992).
Moreover, in vivo inhalation exposure of rodents to acrolein
resulted in inflammation, hyperplasia, and squamous metapla-
sia of the respiratory airway epithelium (Dorman et al., 2008). In
epidemiological studies, an increased lung cancer incidence
was reported for nonsmoking Chinese women cooking in a tra-
ditional wok style. The women exhibited significantly higher
levels of the mercapturic acid metabolites of acrolein, crotonal-
dehyde, and benzene, suggesting a link between lung cancer
and volatile toxicants (such as acrolein) produced by high tem-
perature wok cooking/frying (Wang et al., 2013).
A novel and contradictory hypothesis was recently proposed
suggesting that acrolein may, in fact, be part of a physiological
anticancer system that controls the growth/proliferation of can-
cer cells (Alarcon, 2012). This hypothesis is based on properties
of acrolein such as GSH depletion and direct cytotoxicity, and
the fact that acrolein is a metabolite of anticancer compounds,
spermine and cyclophosphamide. The hypothesis is interesting
but speculative; and needs further investigation. Detailed stud-
ies on the type and magnitude of DNA damage and mutations
arising from endogenous and/or exogenous acrolein exposure,
and the biological consequences are essential to understand the
true role of acrolein in cancer (Figure 2).
Inflammation and Immune Alterations
Adverse health effects of acrolein are anticipated to arise from
its impact on innate and adaptive immune responses.
Inflammation is an essential protective immune response for
combating infections; however, uncontrolled or chronic inflam-
mation is a major cause of tissue injury. Interestingly, reports of
acrolein-induced alterations in inflammatory signaling and
gene expression, particularly via the NF-jB pathway, are diverse
and often conflicting, implying a significant multilevel com-
plexity influenced by cell type, duration of acrolein exposure,
and environmental context (reviewed by Yadav and Ramana
(2013)).
Acrolein can potentially diminish defenses against bacterial
and viral infections by inhibiting macrophage responses/func-
tion via suppression of NF-jB (Horton et al., 1999; Kehrer and
Biswal, 2000; Kirkham et al., 2004; Lambert et al., 2005), or by
direct alkylation of signaling proteins (Hristova et al., 2012;

Acrolein
Carcinogenesis
Disease State
FIG. 2. Effects of acrolein on DNA.
Kasahara et al., 2008; Valacchi et al., 2005), or by suppressing M1
proinflammatory reactions and favoring anti-inflammatory M2
responses, consistent with observations in smokers (Hristova
et al., 2012). Acrolein is shown to suppress TNF-a-induced IL-8, a
proinflammatory neutrophil chemoattractant, in both primary
and immortalized human bronchial epithelial cells (Valacchi
et al., 2005). Immunosuppressive effects of acrolein exposure
were also seen in human T cells, in which acrolein directly alky-
lated Cys-61 and Arg-307 of NF-jB1 (p50), significantly reducing
binding to the promoters of IL-2, IL-10, TNF-a, GMCSF, and IFNc,
with a notable exception being IL-8 (Lambert et al., 2007).
In contrast to these immunosuppressive effects, acrolein is
demonstrated to activate NF-jB and induce proinflammatory
mediators. In rat lung epithelial cells, acute acrolein exposure
(20 mM, 6 h) induced the NF-jB dependent marker of inflamma-
tion COX-2 via calcium release and subsequent proteolytic deg-
radation of IjBa. This was reversed with GW5074, a Ras/Raf-1/
ERK pathway inhibitor (Sarkar and Hayes, 2007). Acrolein
induced an inflammatory response and increased mucin gene
expression in human middle ear epithelial cells supporting the
hypothesis that acrolein may be a risk factor for otitis media
(Song et al., 2013). IL-8 upregulation is associated with many
inflammatory disorders, and IL-8 production due to acrolein
exposure has been reported in vitro in multiple cell types,
including normal human bronchial smooth muscle cells and in
airway epithelial cells (Moretto et al., 2012), alveolar macro-
phages, cultured normal human lung fibroblasts, and in small
airway epithelial cells which was prevented by antioxidants
NAC and MESNA, and by U0126 and FR180204, inhibitors of Akt
and ERK1/2 respectively, suggesting their mechanistic involve-
ment in IL-8 induction (Moretto et al., 2009). Finkelstein showed
acrolein-induced IL-8 production by isolated neutrophils which
was accompanied by inhibition of apoptosis via suppressed cas-
pase activation, extrapolating the potential for prolonged and
increased inflammation within the lung (Finkelstein et al., 2001).
Acrolein-induced IL-8 in human pulmonary fibroblasts was
MOGHE ET AL. | 247
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Altered Gene Expression
effectively suppressed by melatonin (Kim et al., 2012). Similar
proinflammatory effects along with increased susceptibility to
influenza-A virus was seen in vivo wherein mice exposed to
acrolein for 4 days exhibited an accelerated inflammatory
response with higher macrophages and neutrophils, and greater
IL-1a, IL-1b, IL-6, TNF, IFN-c, KC, and MCP-1 in the bronchial
fluid; the effects were suppressed by the carbonyl scavenger
bisulphite (Ong et al., 2012). In vitro acrolein exposure of nasal
epithelial cells was inflammatory and cytotoxic (Comer et al.,
2014). Also, intranasal acrolein exposure of mice caused both
pulmonary inflammation and death of lung epithelial cells,
with increased activated macrophages in the lungs and
increased ROS formation via induction of NF-jB signaling; these
effects were decreased by NAC (Sun et al., 2014).
Recent data indicate that acrolein may contribute to the
highly atherogenic transformation of macrophages into foam
cells via modification of LDL (Watanabe et al., 2013). In keeping
with this, acrolein-lysine adducts in LDL are detected in athero-
sclerotic lesions in humans. Acrolein also increased macro-
phages expression of the atherogenic factors 5-lipoxygenase,
leukotriene B4 and matrix metalloproteinase (Kim et al., 2010;
O’Toole et al., 2009). Other proinflammatory effects of acrolein
include mast cell degranulation which can increase inflamma-
tory injury. Environmentally, relevant exposures of acrolein
(1 ppm) promoted the generation of ROS and initiated
degranulation of the airway mast cells (RBL-2H3); these effects
were significantly reduced by antioxidants NAC and tetrame-
thylthiourea (Hochman et al., 2014). These studies suggest a sig-
nificant role for acrolein in chronic inflammatory disorders via
dysregulation of inflammatory mediators both locally and sys-
temically; however, such conclusions may be premature since
in-vivo studies are limited. Although acrolein appears to have
contradictory pro- and anti-inflammatory effects, they may not
be incongruous. It is likely that the magnitude and duration of
acrolein exposure may critically determine outcomes, wherein
acute high-dose acrolein exposure might suppress innate
 248 | TOXICOLOGICAL SCIENCES, 2015, Vol. 143, No. 2
Acrolein
Acute High
Exposure Dose
Suppression of Immune
Responses
NFkB, TNFα, IL-10, IFNγ, IL-2,
GMCSF
Susceptibility to Infections
FIG. 3. Effects of acrolein on immune responses.
immunity and inflammatory responses thereby increasing sus-
ceptibility to infections, whereas chronic low-dose exposures
may increase inflammation leading to tissue injury (Figure 3).
ER Stress
The ER is the site of synthesis and folding of proteins, and the
accumulation of misfolded proteins can trigger ER stress and
the unfolded protein response (UPR). ER stress is thought to con-
tribute to several pathological conditions that may also be asso-
ciated with increased acrolein, such as fatty liver disease
(Vacaru et al., 2014), Alzheimer’s disease (Li et al., 2014), and
inflammatory and cardiovascular diseases (Zhou and Tabas,
2013). Based on its proclivity to form protein adducts, acrolein is
highly likely to trigger ER stress. Indeed, several recent studies
demonstrate acrolein-induced ER stress and UPR. The effects of
acrolein (2–25 mM) on ER stress and endothelial cell activation
were studied in human umbilical vein endothelial cells
(Haberzettl et al., 2009), wherein both the alarm and the adapta-
tion pathways of UPR were triggered, and inflammatory genes
TNF-a, IL-6, and IL-8 were upregulated, demonstrating the
proinflammatory and atherogenic properties of acrolein.
Notably, the acrolein effects were prevented by the chemical
chaperone (phenylbutyric acid), suggesting its usefulness in
treating pathologies associated with acrolein toxicity. The same
group also demonstrated that the elimination of LPO products
(eg acrolein) by aldose reductase diminishes ER stress and
decreases ischemia-reperfusion injury (Keith et al., 2009). Our
recent work demonstrates that acrolein-induced cytotoxicity in
hepatocytes involves ER stress, in combination with mitochon-
drial disruption and oxidative stress, resulting in cell death
(Mohammad et al., 2012) Acrolein-induced cell death was only
partially attenuated by NAC, phenyl-butyric acid, and caspase
or JNK inhibitors individually, indicating that combination
therapies may be more effective against acrolein-induced
toxicity. Interestingly, acrolein caused ER stress but failed to
upregulate the protective ER-chaperones, GRP78 and GRP94.
Acrolein is demonstrated to modify and reduce function of
another chaperone protein disulfide isomerase; this may lead to
the enhancement and perpetuation of ER stress (Carbone et al.,
2005). Activation of protective versus injurious ER responses to
acrolein exposure may be dependent on dose and time, as
shown in A549 lung cells (Tanel et al., 2014).
In vitro acrolein exposure (<10 mM) of Swiss 3T3 cells
was demonstrated to be primarily responsible for cigarette
Chronic Low
Exposure Dose
Enhancement of Inflammatory
Responses
NFkB, IL-8, COX-2, IL-1β, IL-6,TNF,IFN γ,
KC, MCP-1, 5-lipoxygenase, LTB4, MMP
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Inflammation & Tissue Injury
smoke-induced ER stress and activation of PERK, and ATF4
(Hengstermann and Muller, 2008). In vivo acute (a single intra-
peritoneal injection of acrolein 12 mmol/kg) and chronic (three
weekly intraperitoneal injections of acrolein at 6, 12, or 24 mmol/
kg) systemic acrolein exposure in rats caused ER stress and
upregulation of ATF4, CHOP and GADD34, and VEGF, leading to
emphysematous lung tissue remodeling and airspace enlarge-
ment; caspase-3 activation and apoptosis were concurrently
observed in the septal cells in acrolein-treated rat lungs.
Moreover, accumulated acrolein-protein adducts were observed
in the lungs of smokers with COPD, indicating that acrolein may
contribute to lung disorders like emphysema and COPD
(Kitaguchi et al., 2012). Recent reports have demonstrated ER
stress associated with cigarette smoke and the development of
lung disorders such as emphysema (Gan et al., 2011) and COPD
(Ribeiro and O’Neal, 2012). Thus, acrolein-induced ER stress may
be a relevant mechanism of toxicity in various disorders.
Structural and Membrane Effects
Myelin is a critical structural component of the nervous system
composed of lipids and proteins including myelin basic protein
(MBP), and demyelination is a hallmark of neurodegenerative
diseases, including multiple sclerosis (MS) and SCI. Increased
acrolein levels are seen in the spinal cord in MS and SCI animal
models, with acrolein levels correlating to neuronal membrane
damage, loss of conduction, and even degeneration (Leung et al.,
2011; Shi et al., 2011). The acrolein scavenger hydralazine less-
ened myelin damage and improved behavioral outcomes
(Leunget al., 2011); reversed SCI-induced damage by alleviating
superoxide production, preventing GSH depletion (Hamann
et al., 2008); and effectively reducing acrolein levels, tissue dam-
age, motor deficits, and neuropathic pain (Park et al., 2014).
Acrolein exposure in vitro also elicited a concentration-
dependent modification and aggregation of neurofilament-L
(NF-L) which may contribute to neurodegeneration. The acro-
lein concentrations used (100–500 mM) correspond to concentra-
tions of 1.0–5.2 nmol/mg protein, comparable to levels observed
in Alzheimer’s disease, and thus are within the expected range
of SCI and MS. These effects of acrolein were blocked by NAC
and GSH (Jeong and Kang, 2008).
Acrolein exposures as low as 1 lM for 4 h resulted in
increased permeability of cell membranes to ethidium bromide
in ex vivo spinal cords; as the dose and duration increased,
larger molecules such as horseradish peroxidase and LDH were

able to pass through (Luo and Shi, 2004). Exposure to acrolein
in vivo and in vitro was associated with reduced presynaptic neu-
rotransmitter release; this effect involved inhibition of key -
proteins that regulate membrane-vesicle fusion, such as
N-ethylmaleimide-sensitive fusion protein (NSF) and synapto-
somal-associated protein of 25 kDa (SNAP-25). Acrolein, result-
ing from acrylamide, also inhibited presynaptic membrane
neurotransmitter uptake and vesicular storage in vivo and
in vitro (LoPachin et al., 2004, 2006); proteomic analyses showed
that these dysfunctions were associated with the formation of
acrolein adducts with the dopamine transporter and v-ATPase
(Lopachin et al., 2007).
Acrolein exposure resulted in several structural changes
including induced F-actin microfilament aggregation, and mar-
ginalization and regionalization in Sertoli cells, which are
important in spermatogenesis; such effects may underlie the
testicular toxicity of the anticancer drug cyclophosphamide
which is metabolized into acrolein (Liu et al., 2012). Another
membrane effect of acrolein was the stimulation of erythrocyte
cell membrane scrambling, a typical feature of suicidal death or
eryptosis. Membrane phospholipid scrambling was accompa-
nied by the generation of ceramide, cell shrinkage, and
apoptosis (Ahmed et al., 2013). Airborne acrolein was shown to
induce hyperphosphorylation and damage of keratin-8 and
ubiquitination of intermediate filament in bronchiolar lung cell
monolayers (Burcham et al., 2014). Numerous studies have
shown in vitro and in vivo toxicity of acrolein associated with
structural (and functional) effects in the cardiovascular system,
including increased susceptibility to vasospasm (Conklin et al.,
2006), increased heart rate and blood pressure (Perez et al.,
2013), increased atherosclerotic lesions (Srivastava et al., 2011),
and decreased endothelial cell migration (O’Toole et al., 2014);
however, the exact toxic mechanisms remain unclear.
Deregulated Signal Transduction
Protein kinases and phosphatases are critical components of
intracellular signaling that regulate cellular function, and acro-
lein is reported to inactivate as well as activate them in a con-
textual manner, depending on dose and cell type. Acrolein, at a
fairly high concentrations (100–1000 mM), was shown to be a
potent, time-dependent inactivator of protein tyrosine phos-
phatase-1B via covalent modification of an active site cysteine
(Seiner et al., 2007); and phosphatase PP2A was also inhibited by
acrolein, with concurrent apoptosis (Fathi et al., 2000). On the
other hand, a lower acrolein exposure (25 mM) in human hepato-
cytes led to a modest  2-fold increase in both serine and tyro-
sine phosphatase activity and reduced interferon-a antiviral
activity, which may contribute to therapy resistance in hepatitis
C virus infection (Joshi-Barve et al., 2009). Acrolein exposure
(10–30 mM) in breast cancer cells altered the thioredoxin-
dependent redox system and caused a G2-M cell cycle arrest,
but did not inhibit phosphatase-CDC25 which controls this cell
cycle checkpoint (Beillerot et al., 2012).
Several in vitro studies described below have demonstrated
acrolein-induced alterations in cellular mitogen activated pro-
tein kinases (MAPKs) which are likely to contribute to other
mechanisms of toxicity; however, few findings are validated
in vivo. Acrolein-induced MAPK activation and cytotoxicity were
observed in cultured neurons along with the activation of c-jun
cAMP response element binding protein, CREB (Pugazhenthi
et al., 2006), and in cultured rat vascular smooth muscle cells;
however, in the latter study, MAPK inhibitors failed to block cell
death, suggesting divergent mechanisms (Ranganna et al., 2002).
Activation of p38 in human airway smooth muscle cells and
MOGHE ET AL. | 249
lung fibroblasts exposed to subcytotoxic concentrations of
acrolein (10–100 mM) resulted in the production of a potent
proangiogenic factor, vascular endothelial growth factor (VEGF);
the effects were prevented by p38 inhibitor and NAC (Volpi et al.,
2011). Activation and membrane translocation of another pro-
tein kinase, PKC-d, by allyl alcohol and its primary metabolite
acrolein (up to 125 mM), resulted in cytotoxicity in hepatocytes,
which was blocked by rottlerin, a selective PKC-d inhibitor
(Maddox et al., 2003). Our work showed that acrolein exposure of
cultured hepatocytes led to a sustained activation of ERK1/2,
JNK, and p38, which was associated with ER and mitochondrial
stress and apoptosis; the cytotoxic effects of acrolein were
decreased by JNK inhibitor, suggesting that kinase activation
may be linked to cell death and liver injury (Mohammad et al.,
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2012). Via p38 activation, acrolein also caused upregulation of
the muscle-specific E3 ligases atrogin-1 and MuRF1, suggesting
a possible role in degradation of myosin heavy chain, myotube
catabolism, and muscle wasting (Rom et al., 2013). In contrast,
acrolein was shown to suppress phosphorylation of JNK and
activation of c-Jun; moreover, JNK was a direct target for alkyla-
tion by acrolein at key cysteine residues in the active site
(Hristova et al., 2012).
Acrolein-induced deregulated signaling is known to induce
mucin production and inflammation (major contributors to
lung disorders) via many upstream signals, including (1) MAPKs
and epidermal growth factor receptor (Deshmukh et al., 2005);
(2) matrix metalloproteinase-9 and the Ras/extracellular signal-
regulated kinase pathway, which was attenuated by simvasta-
tin (Chen et al., 2010); (3) Peroxisome proliferator-activated
receptor-gamma (PPAR-gamma), which was reduced by rosigli-
tazone (Liu et al., 2009); and (4) Phosphodiesterase 5 that selec-
tively degrades cyclic guanosine 30 ,50 -monophosphate (cGMP),
which was prevented by sildenafil (Wang et al., 2009). Acrolein
has also been implicated in the demise of retinal pigment epi-
thelium (ARPE-19) cells, with overproduction of the proangio-
genic cytokines, TGFb2 and VEGF that are important in macular
degeneration and in diabetic retinopathy. These effects were
potentiated by high glucose and were attenuated by NBHA
(N-benzylhydroxylamine, a hydroxylamine that sequesters
aldehydes) and by SIS3, a specific inhibitor of SMAD3 that blocks
TGFb2 signaling (Grigsby et al., 2012; Vidro-Kotchan et al., 2011).
Recently, a novel mechanism of acrolein cytotoxicity involving
the acrolein-conjugation and inactivation of glyceraldehyde-
3-phosphate dehydrogenase, an enzyme with many diverse
functions, was described in vitro (Martyniuk et al., 2011) and in
mouse mammary carcinoma FM3A cells (Nakamura et al., 2013).
Thus, acrolein-induced changes in cellular signaling and gene
expression may alter function and physiology, and potentially
contribute to the disease process in multiple organ systems.
CONCLUSIONS AND FUTURE DIRECTON
In this review, we focused on the myriad effects mediated by
acrolein with an emphasis on the molecular mechanisms of
toxicity that provide evidence to suggest that acrolein plays a
critical role in disease pathogenesis (Figure 4). Acrolein operates
via disruption of cell organelles such as mitochondria, ER, and
membranes. In addition, it increases oxidative stress and apop-
tosis, alters proinflammatory mediators, and changes cellular
signal transduction leading to a variety of defects that
may cause or predispose to disease in various organs.
Moreover, acrolein may have cooperative effects with other
toxic insults. Many of the studies described here were done
in vitro or in cultured cells, rather than in vivo; hence, despite
 250 | TOXICOLOGICAL SCIENCES, 2015, Vol. 143, No. 2

MECHANISMS OF TOXICITY AFFECTED ORGANS

DNA
Adduction Brain

Mitochondrial Eye
Dysfunction

Skin
Inflammation
Lung

Oxidative Heart
ACROLEIN Stress
Liver

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Protein Kidney
Adduction

Gut

Membrane Reproductive
Disruption system
Bladder

Endoplasmic
Reticulum Stress

FIG. 4. Toxicity mechanisms and organ systems affected by acrolein (images courtesy Google).

strong links to several diseases, the causal role of acrolein in
disease pathogenesis, though highly likely, can only be sur-
mised. A limitation of this review stems from the fact that levels
of acrolein are difficult to quantify, particularly in vivo, while the
in vitro literature describes acrolein concentrations in various
units that are problematic to interconvert and challenging to
compare.
Several unanswered questions remain regarding acrolein
toxicology. Detailed studies are necessary to determine not only
the temporal sequence of molecular events, but also the inter-
dependence, cross-talk, and the relative contributions of differ-
ent mechanisms of acrolein toxicity to the pathogenesis/
progression of disease. The development of small and large
mammalian models of exogenous and endogenous acrolein
exposures, both acute and chronic, will promote detailed mecha-
nistic investigations and toxicity studies. Research, particularly in
humans, has been limited by availability of techniques to quan-
tify acrolein and acrolein-adducts in body fluids and tissues.
Hence, the development of sensitive, high-throughput and quan-
titative techniques will facilitate large population-based human
studies. Acrolein scavengers with proven effectiveness in small
in vitro/in vivo studies may provide effective and safe means to
mitigate acrolein toxicity and associated disorders; however, their
bioavailability, effective concentrations, and delivery to the
affected areas would require extensive investigations. A true
determination of causality and the contribution of acrolein need
to be established by detailed in vivo studies in animal models and
in human populations. Moreover, it will be important to examine
the impact of age, sex, lifestyle, and genetic variation on the
susceptibility to acrolein exposures in correlation with disease
states.
FUNDING
This work was supported by National Institute on Alcohol
Abuse and Alcoholism (NIAAA) and National Institute of
Environmental Health Sciences (NIEHS) at the National
Institutes of Health (NIH); and U.S. Department of Veterans
Affairs. Grant numbers are: R01AA018869-02 (CJM),
T35ES014559-06 (CJM), P01AA017103-01 (CJM), K01 ES017105-
01A1 (S.J.-B.) and (BX000350 (CJM).
ACKNOWLEDGMENTS
The authors would like to acknowledge Ms Marion McClain
for proof-reading the article. Nothing to disclose.
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