Acrolein and chloroacetaldehyde: An examination of the cell and

cell-free biomarkers of toxicity

Stephanie L. MacAllister, Nicolas Martin-Brisac, Vincent Lau, Kai Yang, Peter J. OBrien

Department of Pharmaceutical Science, Faculty of Pharmacy, University of Toronto, 144 College St., Toronto, Canada M5S 3M2
a r t i c l e i n f o
Article history:
Available online 7 December 2012
Keywords:
Acrolein
Aldehyde dehydrogenase (ALDH)
Chloroacetaldehyde (CAA)
Glutathione (GSH)
Protein carbonylation
Reactive oxygen species (ROS)
a b s t r a c t
Cyclophosphamide and ifosfamide are two commonly used DNA-alkylating agents in cancer chemother-
apy that undergo biotransformation to several toxic and non-toxic metabolites, including acrolein and
chloroacetaldehyde (CAA). Acrolein and CAA toxicities occur by several different mechanisms, including
ROS formation and protein damage (oxidation), however, these pathways of toxicity and protecting
agents used to prevent them have yet to be compared and ranked in a single study. This research focused
on the molecular targets of acrolein and CAA toxicities and strategies to decrease toxicities. Hepatocyte
viability (cytotoxicity) was assessed using Trypan blue uptake; formation of reactive oxygen species
(ROS) and endogenous H
2
O
2
were also assessed in the hepatocyte model. In cell-free models (bovine
serum albumin and hepatic microsomes), protein carbonylation was the measurement of toxicity. The
present study demonstrated that acrolein was a more potent toxin than CAA for freshly isolated rat hepa-
tocytes, bovine serum albumin and rat hepatic microsomes. Acrolein protein carbonylation was depen-
dent on its concentration; as acrolein concentration increased, protein carbonylation increased in a
linear trend, whereas, CAA deviated from the trend and did not cause protein carbonylation at lower con-
centrations (<400 lM). Aldehyde dehydrogenase (ALDH) is a major pathway for detoxifying pathway for
CAA in hepatocytes, as a 3-fold increase in cytotoxicity occurred when cells were incubated with cyan-
amide, an ALDH inhibitor. Inhibiting ALDH or depleting GSH in hepatocytes increased cytotoxicity by
about 3-fold in acrolein-treated hepatocytes. The overall effectiveness of protecting agents to prevent
or suppress acrolein or CAA toxicities in cell and cell-free models were ranked in order of most effective
to least effective: reducing agents (sodium borohydride, sodium bisulte) > thiol-containing compounds
(N-acetylcysteine, cysteine, glutathione, 2-mercaptoethane sulfonate [MESNA], penicillamine) > carbonyl
scavengers/amines (aminoguanidine, hydralazine, hydroxylamine) > antioxidants/ROS scavengers (ascor-
bic acid, Trolox; only utilized in hepatocyte system). An understanding of acrolein and CAA toxicities and
the ability of protecting agents to protect against toxicities may help to establish or improve existing
therapeutic interventions against the side effects associated with acrolein or CAA in cyclophosphamide
or ifosfamide treatment.
2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Cyclophosphamide and ifosfamide are DNA-alkylating agents
commonly used in cancer chemotherapy. Metabolism of these
compounds mainly occurs in the liver, but can also occur in other
sites, including erythrocytes, kidneys and the tumor itself.
Although the metabolic pathways are similar, cyclophosphamide
and ifosfamide differ in the degree of formation of certain metab-
olites. Both compounds are administered as prodrugs that undergo
biotransformation catalyzed by cytochrome P450s. As a byproduct
of this reaction, acrolein is produced [1]. Ifosfamide and, to a lesser
extent, cyclophosphamide are also inactivated by N-dechloroethy-
lation, resulting in N-dechloroethylated metabolites and the
byproduct chloroacetaldehyde (CAA) [1,2]. Although these antican-
cer drugs are widely used for the treatment of a variety of tumor
types, their use is associated with systemic toxicities resulting
from their subsequent metabolism. Acrolein is known to be
responsible for the hemorrhagic cystitis observed in some patients,
whereas CAA is said to be responsible for nephrotoxicity and
neurotoxicity. About 2560% of ifosfamide is metabolized to CAA
and its dechloroethylated metabolites that compete with the 4-
hydroxylation pathway. In contrast, only about 10% of cyclophos-
phamide is dechloroethylated. This could explain the decreased
neuro- and nephrotoxicity side effects of cyclophosphamide as
compared to ifosfamide [1,3].
Acrolein is a highly reactive a,b-unsaturated aldehyde that
readily reacts with cellular nucleophiles, such as the thiol groups
0009-2797/$ - see front matter 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.cbi.2012.11.017

Corresponding author. Tel.: +1 416 978 2716.

E-mail address: peter.obrien@utoronto.ca (P.J. OBrien).
Chemico-Biological Interactions 202 (2013) 259266
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of cysteine residues in proteins and glutathione (GSH), as well as
nitrogen atoms in lysine and histidine groups [4]. Previous litera-
ture suggests that acrolein bladder toxicity can be explained in
three steps: (1) acrolein rapidly enters uroepithelial cells where
it activates intracellular reactive oxygen species (ROS) and NO pro-
duction through iNOS activation, leading to (2) peroxynitrite pro-
duction. Once peroxynitrite is formed, it can lead to (3) damage
to protein, DNA and lipids [5]. Chloroacetaldehyde, like acrolein,
causes GSH depletion at toxic concentrations (above 300 lM)
and partial GSH depletion at sub-toxic concentrations (150
200 lM) in hepatocytes, followed by thiol protein adduct forma-
tion and nally results in lipid peroxidation and cytotoxicity [6].
These cytotoxic byproducts can be detoxied by various aldehyde
dehydrogenases (ALDHs) to their corresponding and less toxic
acids, as well as by conjugation with GSH [1]. Among the metabolic
detoxication pathways, there are several pharmacological ap-
proaches to limit the toxicities of reactive carbonyl species (RCS).
Direct trapping of these compounds, such as the formation of cova-
lent adducts with nucleophilic thiols or direct Schiff bases adducts
with nucleophilic nitrogens, plays an important role in the inhibi-
tion of toxicities, as well as increasing the antioxidant defense sys-
tems against oxidative stress. Compounds possessing these
capabilities include thiols, carbonyl scavengers/amines, and reduc-
ing agents or antioxidants [7].
Acrolein and CAA toxicities occur by several different mecha-
nisms, but extent of toxicity and protection by various chemicals
need to be compared and ranked in a single study. These studies
could provide a better understanding into the therapeutic value
of protective agents currently being prescribed in combination
with ifosfamide and cyclophosphamide treatment, and for other
agents not currently being used, but which have demonstrated
scavenging or protecting effects against acrolein or CAA or other
toxic aldehydes.
2. Materials and methods
2.1. Chemicals
Type II Collagenase was purchased from Worthington (Lake-
wood, NJ). N-2-hydroxyethylpiperazine-N
0
-2-ethanesulfonic acid
(HEPES) was purchased from BoehringerMannheim (Montreal,
Canada). Acrolein, chloroacetaldehyde (CAA), trichloroacetic acid
(TCA), dinitrophenylhydrazine (DNPH), ()-6-hydroxy-2,5,7,8-
tetramethylchromamane-2-carboxylic acid (Trolox), 2-mercaptoe-
thanesulfonate (MESNA) and all other chemicals were obtained
from SigmaAldrich Corp. (Oakville, ON, CAN).
2.2. Animal treatment and hepatocyte preparation
Male SpragueDawley rats weighing 275300 g (Charles River
Laboratories) were used according to the guidelines of the Cana-
dian Council on Animal Care [8]. Hepatocytes were isolated from
rats by collagenase perfusion of the liver as described by Moldus
and colleagues [9]. Isolated hepatocytes (10
6
cells/mL, 10 mL) were
suspended in Krebs-Henseleit buffer containing 12.5 mM HEPES
(free acid) with pH readjusted to 7.4, in rotating 50 mL round-bot-
tomed asks, under an atmosphere of 95% O
2
and 5% CO
2
or 1% O
2
,
94% N
2
and 5% CO
2
in a water bath of 37 C for 30 min prior to the
addition of chemicals.
2.3. Cell viability
Hepatocyte viability was assessed microscopically by plasma
membrane disruption as determined by the Trypan blue (0.1% w/
v) exclusion test [9]. Hepatocyte viability was determined every
30 min during a 3 h incubation period. Hepatocytes used were
8090% viable before use. GSH-depleted hepatocytes were ob-
tained by preincubating hepatocytes with 200 lM 1-bromohep-
tane for 30 min prior to the addition of other agents [10].
Aldehyde dehydrogenase (ALDH)-inhibited hepatocytes were ob-
tained by preincubating cells with 200 lM cyanamide for 45 min.
The concentrations of inhibitors/modulators used were nontoxic.
2.4. Microsomal preparation
Adult male SpragueDawley rats (250300 g) were anesthe-
tized by sodium pentobarbital (60 mg/kg body). Livers were re-
moved under sterile conditions and perfused with KCl solution
(1.18% w/v, 4 C). Hepatic microsomes were prepared by differen-
tial centrifugation as described by Dallner et al. [11]. The micro-
somal pellet was suspended and homogenized in sterile 50 mM
potassium phosphate buffer with 0.23% (w/v) KCl pH 7.4 before
storage at 80 C. Microsomal protein was determined by the
method of Joly et al. [12].
2.5. Protein carbonylation assay
Bovine serum albumin or microsome suspensions were either
treated simultaneously with acrolein or CAA and the therapeutic
agents, or the suspensions were pre-treated with acrolein or CAA
for 1 h prior to the addition of the protecting agents for the anti-
dotal effect. Time zero of the reaction began with the addition of
acrolein or CAA. The total protein-bound carbonyl content was
measured by derivatizing the protein carbonyl adducts with 2,4-
dinitrophenylhydrazine (DNPH). An aliquot of hepatocytes, bovine
serum albumin (2 mg/mL or 30 lM in 0.1 M sodium phosphate
buffer, pH 7.4) or microsomal suspension (0.5 mL) at different time
points was added to an equivalent volume (0.5 mL) of 0.1% DNPH
(w/v) in 2.0 N HCl and allowed to incubate for 1 h at room temper-
ature. The reaction was terminated and the total cellular protein
precipitated by the addition of 1.0 mL volume of TCA (20% w/v).
Protein was pelleted by centrifugation at 5000g for 1 min, and
the supernatant was discarded. Excess unincorporated DNPH was
extracted three times using an excess volume (0.5 mL) of etha-
nol:ethyl acetate (1:1) solution. Following extraction, the recov-
ered cellular protein was dried under a stream of nitrogen and
dissolved in 1 mL of 2 M Tris-buffered 8.0 M guanidineHCl, pH
7.2. The resulting solubilized hydrazones formed were measured
using a SpectraMax Plus384 spectrophotometer at 370 nm. The
concentration of DNPH derivatized protein carbonyls dissolved in
guanidine-HCl was determined using an extinction coefcient of
22,000 M
1
cm
1
[13].
2.6. Reactive oxygen species (ROS) formation
Hepatocyte ROS formation was determined by adding dichloro-
uorescein diacetate (DCFH-DA) to the hepatocyte incubate. DCFH-
DA penetrates hepatocytes and is hydrolyzed to form a non-uo-
rescent dichlorouorescein (DCF). DCF then reacts with ROS to
form the highly uorescent dichlorouorescein and efuxes the
cell. ROS formation was assayed by withdrawing 1 mL samples,
which were then centrifuged for 1 min at 5000g. The cells were
resuspended in 1 mL Krebs-Heinseleit buffer containing 2 lM
DCFH-DA and incubated at 37 C for 10 min, and the uorescence
intensity was measured using a SpectraMax Gemini XS uorimeter
at 490 nm excitation and 520 nm emission [14].
2.7. Hepatocyte H
2
O
2
measurement
H
2
O
2
was measured in hepatocytes by taking aliquots at 30 or
90 min using the FOX 1 reagent (ferrous oxidation of xylenol
260 S.L. MacAllister et al. / Chemico-Biological Interactions 202 (2013) 259266
orange). The FOX 1 reagent consisted of 25 mM sulfuric acid,
250 lM ferrous ammonium sulfate, 100 lM xylenol orange and
0.1 M sorbitol. At the given time intervals 50 lL of hepatocyte sus-
pension (10
6
cells/mL) were added to 950 lL FOX 1 reagent and
incubated for 30 min at room temperature. The absorbance of the
samples were read at 560 nm and the concentration of H
2
O
2
was
determined using the extinction coefcient of 2.67 10
5
M
1
-
cm
1
[15,16].
2.8. Statistical analysis
Statistical analysis was performed by a one-way ANOVA (anal-
ysis of variance) test, and employing Tukeys posthoc test to assess
signicance.
3. Results
3.1. Prevention of carbonylation of bovine serum albumin caused by
acrolein and CAA
The damage that acrolein and CAA could cause to protein was
investigated by incubating each aldehyde with bovine serum albu-
min for 60 min and measuring the level of protein carbonylation by
using DNPH. An increase in bovine serum albumin protein carbon-
ylation occurred when increasing concentrations of acrolein or CAA
were present (Fig. 1). Based on the bovine serum albumin carbon-
ylation concentrations listed in Table 1, at 2 mg/mL or 30 lM initial
concentration of bovine serum albumin, about 12 amino acid
groups per molecule were modied by acrolein or CAA. A signi-
cant increase in protein carbonylation was observed in bovine ser-
um albumin when as low as 50 lM of acrolein was incubated.
Based on Fig. 1, protein carbonylation appears to be dependent
upon acrolein concentration, whereas the protein carbonylation
caused by CAA does not occur at lower concentrations (<400 lM)
and requires higher concentrations induce protein carbonylation.
The effectiveness of protecting agents against protein carbonyl-
ation induced by acrolein and CAA at 120 min was examined by
measuring the DNPH derivatized protein carbonyl content. Ali-
quots were measured at 120 min as this was the peak concentra-
tion in protein carbonylation observed throughout a 3 h
incubation. In order to produce large extents of protein carbonyl-
ation and to examine the capacity of protective agents (thiols, car-
bonyl scavengers/amines and reducing agents), higher
concentrations of acrolein (2 mM) and CAA (4 mM) were used, as
reported in Table 1. The concentrations of all protecting agents
were equimolar to the concentrations of acrolein or CAA.
The most effective agents for preventing carbonylation of bo-
vine serum albumin by both acrolein and CAA were the reducing
agents sodium borohydride and sodium bisulte, which can react
stoichiometrically with the toxins (Table 1). The thiol-containing
compounds, more specically, N-acetylcysteine, cysteine, GSH,
MESNA, were effective at preventing both acrolein and CAA protein
carbonylation with bovine serum albumin, with cysteine being as
protective as the reducing agents, whereas penicillamine was only
protective against acrolein-induced protein carbonylation. The car-
bonyl scavengers/amines were generally more protective against
protein carbonylation by CAA than by acrolein. Hydralazine and
hydroxylamine were almost as effective as thiols for protection
against CAA-induced protein carbonylation.
The antidotal effect occurred when the bovine serum albumin
suspension was treated with acrolein or CAA for 1 h prior to the
addition of the protecting agents (thiols, carbonyl scavengers/
amines and reducing agents); this effect was studied to determine
whether the toxicity occurring was reversible. In general, the anti-
dotal treatment was less effective than the simultaneous protocol
(Table 1). None of the protecting agents, with the exception of so-
dium borohydride and sodium bisulte, substantially reversed car-
bonylation. The antidotal effects with the protecting agents were
smaller against acrolein than against CAA. Thiols, in particular
MESNA, and the carbonyl scavenger hydralazine signicantly pro-
tected against CAA-induced bovine serum albumin protein carbon-
ylation, while penicillamine and aminoguanidine were the least
effective.
3.2. Protection of microsomal protein carbonylation induced by
acrolein and CAA
The study of protein carbonylation was extended to a more
complex system by treating hepatic microsomes with each alde-
hyde and testing for protective antidotal effects. Acrolein caused
protein carbonylation in microsomes to a slightly less extent than
in bovine serum albumin, but the extent was similar for the reac-
tion with CAA. This could be due to the more complex microsomal
system and the ability of acrolein or CAA to generate other types of
toxicities, such as lipid peroxidation that could compete with reac-
tions with proteins. The microsome protein carbonylation results
Fig. 1. Concentration-dependent protein carbonylation of bovine serum albumin by acrolein and CAA treatment. Measurement was taken at 60 min. Baseline corrected by
subtracting control (5.11 0.21 lM) Means SE for three separate experiments are given. Bovine serum albumin: 2 mg/mL in 0.1 M sodium phosphate buffer solution, pH 7.4

signicant as compared to control (P < 0.05).

S.L. MacAllister et al. / Chemico-Biological Interactions 202 (2013) 259266 261
demonstrated that, aside from sodium borohydride, the thiols col-
lectively were the most effective protecting agents (Table 1).
Treating the microsomes with acrolein or CAA for 1 h prior to
the addition of the protecting agents established the antidote capa-
bilities of these agents against protein carbonylation (Table 1). The
protecting agents were generally less effective as an antidote
against protein carbonylation in the microsomal system as com-
pared to bovine serum albumin. Unfortunately, none of the pro-
tecting agents were effective as antidotes against acrolein protein
carbonylation in microsomes with the exception of sodium boro-
hydride, sodium bisulte, hydralazine and hydroxylamine, which
demonstrated a statistically signicant decrease in protein carbon-
ylation as compared to acrolein-treated microsomes. However, all
of the thiols, in addition to sodium borohydride, sodium bisulte
and aminoguanidine were the most effective antidotes against
CAA protein carbonylation in microsomes. In fact, all the antidotes,
with the exception of hydroxylamine, demonstrated a signicant
decrease in CAA-induced protein carbonylation as compared to
CAA-treated microsomes.
3.3. Acrolein or CAA cytotoxicity in highly oxygenated (95% O
2
) versus
reduced (1% O
2
) environment
Freshly isolated rat hepatocytes were incubated under an atmo-
sphere of 95% O
2
or 1% O
2
, and cytotoxicity was determined by the
Trypan blue exclusion test every hour during a 3 h incubation.
Fig. 2 demonstrates a general trend of decreased cytotoxicity of
both acrolein- and CAA-treated hepatocytes in a reduced oxygen
environment at 120 min of reaction. At 75 and 150 lM acrolein
(Fig. 2A), cytotoxicity was signicantly lower in 1% O
2
than in
95% O
2
. Likewise, CAA toxicity (Fig. 2B) at 125 and 250 lM was sta-
tistically signicantly lower in 1% O
2
than in 95% O
2
at 120 min.
3.4. Comparison of the inhibition of detoxication pathways of
acrolein and CAA in hepatocytes
Freshly isolated rat hepatocytes were incubated under an atmo-
sphere of 95% O
2
and 5% CO
2
with acrolein or CAA and inhibitors of
their detoxication, and cytotoxicity was determined by the Try-
pan blue exclusion test every hour during a 3 h incubation. As
shown in Fig. 3, hepatocytes treated with inhibitors of detoxifying
pathways resulted in an increase in hepatocyte cytotoxicity. The
alcohol aversion therapy drug, cyanamide (active component in
Temposil and Dipsan) was used to inhibit ALDH in acrolein- and
CAA-treated hepatocytes by preincubating hepatocytes with cyan-
amide (200 lM) for 45 min prior to other agents. Inhibition of the
ALDH detoxifying pathway by cyanamide and depletion of GSH
with 1-bromoheptane caused 100% cell death in 2 h with acro-
lein-treated cells, with an approximate 3-fold increase in toxicity
as compared to acrolein-treated hepatocytes with both ALDH
Table 1
Acrolein- and chloroacetaldehyde-induced protein carbonylation in cell-free systems of bovine serum albumin or hepatic microsomes.
Treatment BSA Protein
Carbonylation (lM)
BSA Protein Carbonylation
(lM) (antidote effect)
Microsome Protein
Carbonylation (lM)
Microsome Protein Carbonylation
(lM) (antidote effect)
120 min 120 min 120 min 120 min
Control (untreated) 4.9 0.2 4.9 0.2 5.7 0.1 5.7 0.1
2 mM Acrolein 54.9 5.8
a
54.9 5.8
a
43.0 2.5
a
43.0 2.5
a
Thiol Group:
+2 mM N-Acetyl-L-cysteine 23.5 0.1
b
45.5 4.2
a
12.9 0.1
a
39.7 2.7
a
+2 mM L-Cysteine 6.3 2.2
a
27.0 2.1
a
14.2 0.3
a
36.5 2.5
a
+2 mM Glutathione 22.3 3.5
a
36.1 1.6
a
21.2 0.1
a,a
40.3 4.2
a
+2 mM MESNA 12.3 0.1
a
35.0 3.2
a
18.9 0.1
a,a
36.9 3.2
a
+2 mM Penicillamine 11.1 6.2
a
41.2 4.2
a
17.2 0.1
a,a
41.1 3.7
a
Carbonyl scavengers/amines
+2 mM Aminoguanidine 32.6 0.8
a
36.2 2.5
a
31.0 2.2
a,a
36.7 1.2
a
+2 mM Hydralazine 18.8 0.3
a
30.2 3.7
a
17.1 0.9
a,a
28.1 0.7
a,a
+2 mM Hydroxylamine 15.4 1.3
a
34.6 1.6
a
18.9 0.1
a,a
27.5 1.3
a,a
Reducing agents
+2 mM Sodium Borohydride 6.2 0.1
a
9.2 2.1
a
7.0 0.8
a
5.64 0.04
a
+2 mM Sodium Bisulte 8.4 0.7
a
8.6 0.5
a
16.5 0.5
a,a
16.9 2.0
a,a
4 mM CAA 43.5 9.6
a
43.5 9.6
a
41.1 1.2
a
41.1 1.2
a
Thiol Group
+4 mM N-Acetyl-L-cysteine 6.1 2.7
c
15.7 1.2
c
19.1 0.2
a,c
20.0 0.4
a,c
+4 mM L-Cysteine 9.3 2.2
c
22.6 3.2
c
16.0 1.3
c
15.4 0.1
a,c
+4 mM Glutathione 6.1 2.7
c
19.3 1.7
c
13.5 0.7
c
20.8 0.3
a,c
+4 mM MESNA 6.7 0.3
c
10.9 1.4
c
18.3 0.1
a,c
18.9 0.3
a,c
+4 mM Penicillamine 45.2 2.0
a
41.4 5.2
a
16.3 0.1
a,c
21.4 0.2
a,c
Carbonyl scavengers/amines
+4 mM Aminoguanidine 19.1 2.2
c
41.1 2.3
a
33.3 3.2
a
15.8 0.3
a,c
+4 mM Hydralazine 9.8 0.9
c
21.5 2.4
c
24.1 0.3
a,c
21.2 0.3
a,c
+4 mM Hydroxylamine 10.0 2.1
c
30.6 2.1
a
34.5 6.1
a
44.5 2.0
a
Reducing agents
+4 mM Sodium Borohydride 4.4 0.6
c
4.8 0.7
c
6.5 0.3
c
6.1 0.1
c
+4 mM Sodium Bisulte 5.7 1.2
c
5.9 1.0
c
10.9 0.5
a,c
8.48 0.03
c
BSA or microsome suspensions were either treated simultaneously with acrolein or CAA and the therapeutic agents, or the suspensions were pre-treated with acrolein or CAA
for 1 h prior to the addition of the protecting agents for the antidotal effect ().
CAA, Chloroacetaldehyde; MESNA, 2-Mercaptoethane sulfonate.
Bovine serum albumin, BSA: 2 mg/mL in 0.1 M sodium phosphate buffer solution, pH 7.4.
Microsomes: 2 mg/mL in 0.1 M sodium phosphate buffer solution, pH 7.4.
Means SE for three separate experiments are given.
a
signicant as compared to control (P < 0.05).
b
signicant as compared to 2 mM acrolein (P < 0.05).
c
signicant as compared to 4 mM CAA (P < 0.05).
262 S.L. MacAllister et al. / Chemico-Biological Interactions 202 (2013) 259266
inhibited and GSH-depleted hepatocytes. Inhibition of ALDH
caused the greatest increase in cytotoxicity with a 3-fold increase
with CAA-treated hepatocytes. Although depleting GSH in CAA-
treated hepatocytes increased cytotoxicity, this increase was not
signicant. All modulating chemicals had no signicant effect on
hepatocyte cytotoxicity.
3.5. The effect of protecting agents on acrolein- and CAA-induced
hepatocyte cytotoxicity
Freshly isolated rat hepatocytes were incubated under an atmo-
sphere of 95% O
2
and 5% CO
2
, and cytotoxicity and the effects of
protecting agents (1 mM) on acrolein- and CAA-induced cytotoxic-
ity were determined by the Trypan blue exclusion test every hour
for 3 h. Additionally, ROS formation using DCFH-DA and H
2
O
2
mea-
surement using FOX 1 reagent were also assessed at 30 min. The
thiol-containing compounds were the most effective protecting
agents at preventing acrolein cytotoxicity (Table 2). The reducing
agents were also effective in preventing acrolein-induced cell
death, whereas the antioxidants and ROS scavengers, ascorbic acid
and Trolox, were the least effective in preventing acrolein cytotox-
icity. The ROS scavenger, ascorbic acid, was unable to prevent acro-
lein-induced cytotoxicity, however, lower concentrations (300 lM)
of ascorbic acid did delay cytotoxicity at 60 min (results not
shown), suggesting that ascorbic acid becomes pro-oxidant at
higher concentrations; this is also evident with the signicant in-
crease in H
2
O
2
levels with both acrolein- and CAA-treated hepato-
cytes. In contrast, Trolox, delayed cytotoxicity at 60 min for
acrolein treated hepatocytes, but it became ineffective at prevent-
ing cytotoxicity at 120 and 180 min. With CAA-treated hepato-
cytes, Trolox was able to prevent CAA-cytotoxicity up to 180 min.
Hydroxylamine was not effective at preventing acrolein- or CAA-
Fig. 2. Hepatocyte cytotoxicity comparison of (A) acrolein and (B) CAA in 95% or 1%
O
2
levels (120 min). CAA, Chloroacetaldehyde. Means SE for three separate
experiments are given.

signicant as compared to control (P < 0.05).

signicant
within concentration group (P < 0.05).
Fig. 3. Modulation of (A) acrolein- and (B) CAA-induced cytotoxicity by ALDHinhibitor cyanamide or GSH-depleted hepatocytes. GSH-depleted hepatocytes were prepared by
pre-incubation of hepatocytes for 30 min with 200 lM 1-bromoheptane before the addition of other agents. ALDH-inhibited hepatocytes were prepared by pre-incubation of
hepatocytes for 45 min with 200 lM cyanamide before the addition of other agents. Means SE for three separate experiments are given.
a
signicant as compared to control
hepatocytes (P < 0.05).
b
signicant as compared to 75 lM acrolein (P < 0.05).
c
signicant as compared to 125 lM CAA (P < 0.05).
S.L. MacAllister et al. / Chemico-Biological Interactions 202 (2013) 259266 263
induced toxicity in hepatocytes. In fact, hydroxylamine signi-
cantly increased endogenous H
2
O
2
, as assessed by FOX 1 reagent.
4. Discussion
The toxic effects observed with cyclophosphamide and ifosfa-
mide treatment have been attributed to their metabolites, acrolein
and CAA, both formed as byproducts of the major and minor met-
abolic pathways of these agents. This study examined the interac-
tions between acrolein or CAA and various protecting agents in cell
and cell-free models. The protecting agents chosen in this study
were based upon previous literature that has demonstrated scav-
enging or protecting effects against acrolein or CAA or other toxic
aldehydes. The research was focused on the protecting agents abil-
ity to prevent toxicities related to acrolein or CAA, namely protein
carbonylation and ROS formation. A comprehensive understanding
of the ability of certain protecting agents (thiols, carbonyl scaveng-
ers/amines, and reducing agents) to prevent acrolein or CAA toxic-
ities against specic molecular targets may help to establish or
improve existing therapeutic interventions against the side effects
associated with acrolein or CAA.
In this study, acrolein was more reactive with proteins and in-
duced protein carbonylation in bovine serum albumin or micro-
some suspensions at much lower concentrations as compared to
CAA. Table 1 demonstrates that bovine serum albumin, at 2 mg/
mL or 30 lM concentration, had about 12 amino acid groups
per molecule that were modied by acrolein or CAA. Acrolein has
two reactive moieties contributing to its high reactivity towards
biological nucleophiles, which results in toxicity. Acrolein is able
to undergo Michael addition; this type of reaction is common with
thiol groups. Acrolein can also directly form a Schiff base with ami-
no groups. The Schiff base formation is less facile as compared to
the Michael addition, but it can be subsequently accelerated after
the Michael addition reaction has occurred [7,17]. CAA also has
two potential sites of reactivity with biological nucleophiles; in a
typical S
N
2-type reaction, the a-chlorine can be displaced or the
carbonyl group can undergo reactions to form both reversible
and irreversible adducts [6].
Cytotoxicity occurred in both oxidative and reduced environ-
ments for both acrolein and CAA; however, for both toxins, the oxi-
dative environment further augmented cytotoxicity. This suggests
that both compounds, acrolein and CAA, involve oxidative stress-
mediated toxicities in this hepatocyte model.
ALDH can oxidize acrolein and CAA to their corresponding and
less toxic carboxylic acids, acrylic acid and chloroacetic acid,
respectively. Glutathione is an endogenous antioxidant that is
capable of conjugating with electrophiles, such as acrolein and
CAA; this is another detoxifying pathway of acrolein and CAA [1].
Table 2
Hepatocyte protection of acrolein- or CAA-induced cytotoxicity by carbonyl-trapping agents, thiol groups, reducing agents and antioxidants.
Treatment Cytotoxicity (% Trypan Blue Uptake) ROS Formation (%) Endogenous H
2
O
2
Measurement
(nmoles/10
6
cells)
60 min 120 min 180 min 30 min 30 min
Control 19 1 22 2 24 2 100 6.8 0.3
100 lM Acrolein 60 3
a
75 3
a
83 4
a
203 5
a
14.6 1.2
a
Thiol Groups
+1 mM N-Acetyl-L-cysteine 26 2
b
30 1
b
37 2
b
140 3
a,b
6.4 0.7
b
+1 mM L-Cysteine 25 1
b
29 2
b
34 2
b
132 2
a,b
6.3 0.5
b
+1 mM MESNA 27 2
b
34 2
b
39 3
b
128 3
a,b
6.7 0.7
b
+1 mM Penicillamine 49 2
a
55 2
a,b
60 2
a,b
116 2
b
10.9 0.9
Carbonyl scavengers/amines
+1 mM Aminoguanidine 69 5
a
78 6
a
80 4
a
134 5
a,b
8.8 0.7
+1 mM Hydralazine 53 3
a,b
72 3
a
77 3
a
127 3
a,b
7.6 0.9
b
+1 mM Hydroxylamine 78 4
a,b
79 3
a
85 5
a
190 5
a
20.4 1.9
a
Reducing Agents
+1 mM Sodium Borohydride 36 3
a,b
62 2
a
68 2
a
145 3
a,b
9.0 0.9
+1 mM Sodium Bisulte 39 2
a,b
52 2
a,b
57 2
a,b
132 2
a,b
7.1 0.3
b
Antioxidants/ROS scavengers
+1 mM Ascorbic acid 63 4
a
77 4
a
88 6
a
124 6
a,b
21 3
a,b
+1 mM Trolox 40 2
a,b
73 3
a
85 4
a
85 1
b
6.7 0.3
b
300 lM CAA 57 2
a
76 3
a
91 3
a
150 2
a
12.0 0.6
Thiol Groups
+1 mM N-Acetyl-L-cysteine 30 2
c
44 1
a,c
65 2
a,c
107 4
c
8.2 0.3
+1 mM L-Cysteine 30 1
c
53 2
a,c
59 2
a,c
147 5
a
11.3 2.2
+1 mM MESNA 32 2
c
55 2
a,c
68 3
a,c
105 3
c
7.0 0.3
+1 mM Penicillamine 47 2
a
66 2
a
89 2
a
141 2
a
7.3 0.3
Carbonyl scavengers/amines
+1 mM Aminoguanidine 52 5
a
72 6
a
91 4
a
154 6
a
7.3 1.3
+1 mM Hydralazine 61 3
a
87 3
a
92 4
a
119 1
c
8.3 0.7
+1 mM Hydroxylamine 85 4
a,c
89 3
a
93 5
a
272 13
a,c
14.1 1.0
Reducing agents
+1 mM Sodium Borohydride 47 2
a
53 3
a,c
56 2
a,c
118 2
a
5.7 0.5
+1 mM Sodium Bisulte 40 2
a,c
46 2
a,c
48 3
a,c
117 3
a
8.1 0.8
Antioxidants/ROS scavengers
+1 mM Ascorbic acid 55 2
a
63 2
a
91 2
a
184 11
a,c
23.7 4.3
a,c
+1 mM Trolox 38 1
a,c
42 2
a,c
52 1
a,c
134 4
a
8.7 1.2
CAA, Chloroacetaldehyde; MESNA, 2-Mercaptoethane sulfonate.
Means SE for three separate experiments are given.
a
signicant as compared to control (P < 0.05).
b
signicant as compared to 100 lM acrolein (P < 0.05).
c
signicant as compared to 300 lM CAA (P < 0.05).
264 S.L. MacAllister et al. / Chemico-Biological Interactions 202 (2013) 259266
As shown in Fig. 3A, toxicity from 75 lM acrolein was greatly in-
creased when acrolein detoxifying pathways were inhibited. Inhi-
bition of ALDH or depletion of GSH caused signicant and similar
increases in cytotoxicity in acrolein-treated hepatocytes. In con-
trast, the results in Fig. 3B show that the alcohol aversion therapy
drug, cyanamide (active component in Temposil and Dipsan), had a
larger effect on cytotoxicity, with a 3-fold increase in toxicity with
CAA-treated hepatocytes as compared to GSH depleted hepato-
cytes. These results suggest that ALDH plays larger role in detoxi-
fying CAA in this hepatocyte model.
The most effective protecting agents at preventing acrolein and
CAA toxicity with respect to protein carbonylation, cytotoxicity,
and ROS formation, were the reducing agents sodium borohydride
and sodium bisulte. Sodium borohydride is used to reduce car-
bonyl groups, such as ketones and aldehydes, as well as to reduce
Schiff bases, whereas sodium bisulte forms a bisulte adduct with
aldehyde groups. Sodium bisulte can also act as an antioxidant, as
sultes, which can occur naturally in foods, are often used as an
additive to prevent nonenzymatic and enzymatic browning
(caused by oxidation) or for antimicrobial preservative effects
[18,19]. None of the protecting agents were effective as antidote
against protein carbonylation by acrolein or CAA, except for so-
dium borohydride and sodium bisulte (Table 1). This suggests
that protein carbonylation is reversible or that the carbonyl groups
are scavenged. However, the thiol compounds and the carbonyl
scavenging/amine agents used were unable to reverse the damage
caused by acrolein or CAA.
The reactivity of thiols with acrolein or CAA makes them good
candidates for aldehyde detoxication by forming stable adducts
through a nucleophilic reaction [7]. The thiol moiety acts as a
nucleophile, displacing the chloride of CAA or through Michael
addition with acrolein. Of the thiols, cysteine was the most effec-
tive at preventing hepatocyte cytotoxicity, decreasing the endoge-
nous H
2
O
2
, and preventing bovine serum albumin protein
carbonylation. The high reactivity of cysteine has been attributed
to the close vicinity of the amino group on the cysteine residue.
Cysteine reacts through Michael addition with acrolein, this in-
creases the reactivity of the carbonyl group, which can then either
react with a nearby amino group to form a Schiff base, or the
monoadduct can react with an additional cysteine molecule to
form a thiazolidine ring, this however, cannot occur with GSH
[7,17]. Both cysteine and N-acetylcysteine are analogs of GSH. N-
acetylcysteine was also effective at preventing both acrolein and
CAA toxicity (Table 2) and preventing protein carbonylation (Ta-
ble 1). N-acetylcysteine is currently utilized in the clinic to treat
acetaminophen overdose [20]. It has also been previously demon-
strated to have a protective effect against acrolein toxicity in cells
[21,22]. MESNA was another effective agent at preventing acrolein
and CAA toxicities in both cell and cell-free systems. MESNA is co-
administered to patients currently being prescribed cyclophospha-
mide or ifosfamide in order to prevent hemorrhagic cystitis caused
by acrolein. Previous studies have shown that MESNA can react
with CAA and has also been shown to reduce CAA in vitro toxicity,
however, in vivo, MESNA cannot reduce CAA nephrotoxicity
[3,23,24]. Penicillamine was somewhat effective at preventing
acrolein cytotoxicity in hepatocytes; however, it was not effective
against CAA. Penicillamine is used as a copper chelator for the
treatment of Wilsons disease and rheumatoid arthritis; it is known
to scavenge carbonyls. Previous literature has shown penicillamine
scavenging other toxic aldehyde by forming a thiazolidine com-
pound with the aldehyde moiety [25,26].
Compared to thiol-containing compounds, carbonyl scavengers/
amines were overall less effective in preventing both acrolein and
CAA toxicities. The amino group of the carbonyl scavengers acts as
a nucleophile forming a Schiff base and Michael addition product
with acrolein or in a typical S
N
2-type reaction, displacing the a-
chlorine of CAA. Hydralazine was effective at reducing protein car-
bonylation in the cell-free systems; however, it was not an effec-
tive protecting agent in the hepatocytes. Hydroxylamine and its
derivatives have been used previously to sequester aldehydes,
forming oxime compounds, which have been used successfully
in vivo and in vitro to sequester acrolein and prevent its toxic ef-
fects in models of murine neurodegeneration and in cultured
mouse mammary carcinoma cells [27,21]. Unfortunately, hydrox-
ylamine was not effective at sequestering acrolein; in fact, it signif-
icantly increased the H
2
O
2
measurement as assessed by FOX 1
reagent. This likely occurred due to hydroxylamines ability to
act as a catalase inhibitor, which is involved in the decomposition
of H
2
O
2
to H
2
O [28].
The antioxidant and ROS scavenging abilities of ascorbic acid
(vitamin C) and Trolox, a water-soluble analog of a-tocopherol,
were compared in hepatocytes. Although both compounds possess
antioxidant capabilities, both can also exhibit pro-oxidant proper-
ties [29]. Ascorbic acid is an important water-soluble antioxidant
that reduces sulfhydryl groups, scavenges free radicals, and pro-
tects against endogenous oxidative DNA damage [30]. It has also
been identied that ascorbic acid can undergo a Michael-type
addition to acrolein [31]. Ascorbic acid, was unable to prevent
acrolein-induced cytotoxicity, however, lower concentrations
(300 lM) of ascorbic acid did delay cytotoxicity at 60 min (results
not shown), suggesting that ascorbic acid becomes pro-oxidant at
higher concentrations; this is also evident with the signicant in-
crease in H
2
O
2
levels with both acrolein- and CAA-treated hepato-
cytes. In contrast, Trolox, delayed cytotoxicity at 60 min for
acrolein treated hepatocytes, however, it became less effective at
preventing cytotoxicity at 120 and 180 min, while with CAA-trea-
ted hepatocytes, Trolox was able to prevent CAA-cytotoxicity up to
180 min. However, Trolox was more effective at preventing cyto-
toxicity induced by CAA, but only delayed cytotoxicity with acro-
lein-treated hepatocytes.
Utilizing cell and cell-free systems, the overall effectiveness of
protecting agents in order of most effective to least effective were
reducing agents > thiols > carbonyl scavengers/amines > antioxi-
dants/ROS scavengers (only utilized in hepatocyte system).
Conict of Interest
The authors declare that there are no conicts of interest.
Acknowledgements
This research has been funded by the Natural Sciences and Engi-
neering Research Council of Canada Grant.
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