American Journal of Anal yt ical Chemistry, 2011, 2, 832-839
doi:10.4236/ajac.2011.27095 Published Online November 2011 (http://www.SciRP.org/journal/ajac)
Role of Polyamines in Ozone Exposed
Ischemic-Reperfused Hearts*
Rajat Sethi1#, Sai Raghuveer Chava2, Sajid Bashir2,3†, Mauro E. Castro2#
1Cardiovascular Research and Development Laboratory, Department of Pharmaceutical Sciences, Rangel College of
Pharmacy, Texas A & M Health Science Center, Kingsville, Texas, USA
2Department of Chemistry, Texas A & M University-Kingsville, Kingsville, Texas, USA
3Chemical Biol ogy Research Group (CBRG), Kingsville, Texas, USA
Received August 6, 2011; revised September 15, 2011; accepted September 24, 2011
Abstract
The effect of chronic ozone exposure to ischemia reperfusion (I/R) injury in isolated perfused rat hearts was
previously demonstrated. The present study tested our hypothesis that chronic ozone exposure led to attenua-
tion of polyamines in the heart, which may limit sensitivity to I/R. Sprague Dawley rats were continuously
exposed for 8 hrs/day for 28 days to filtered air or 0.8 ppm ozone. Isolated hearts were previously subjected
to 0.5 hour of global ischemia followed by 1 hour of reperfusion after which global polyamine content was
examined between the two groups. Spermidine production was significantly increased in the experimental
group compared to control group (of I/R hearts). These results suggest that ozone-induced sensitivity to
chronic I/R injury activates myocardial polyamine stress response characterized by increased enzymatic ac-
tivities and accumulation of spermidine. Collectively, these results suggest that I/R possibly disturbs poly-
amine metabolism, and increased oxidative stress and concomitant reduced myocardial cell viability.
Keywords: Environmental Pollutants, Cardiovascular Disease, Polyamines, Ischemia-Reperfusion (I/R) In-
jury, Oxidative Stress
1. Introduction
Cardiovascular disease is one of the leading causes of
mortality in the United States with 81 million reported
cases from cardiovascular disease (CVD) in 2006 [1]. Risk
factors for CVD are high blood pressure and coronary
heart disease (myocardial infarction), which is acute heart
attack and angina pectoris (chest pain). Environmental
pollutants play an important role in heart disease, as dem-
onstrated by our previous study [2]. Ozone, has been
shown to increase heart disease, possibly through oxida-
tion of the plasma membrane resulting in apoptosis of
cardiomyocytes [3] or ozone-induced inflammatory re-
sponse (mediators released into the circulatory system [4])
resulting in organ damage and resultant CVD. Although,
the effect of chronic exposure of ozone on CVD was pre-
viously investigated and shown to be related to changes in
cardiac function after I/R injury. This approach measured
left ventricular end diastolic pressure (LVEDP) which
decreased whereas myocardial tumor necrosis factor-alpha
(TNF alpha) and lipid peroxidation levels increased. In
addition, superoxide dismutase (SOD) and IL-10 levels
decreased in ozone exposed I/R hearts compared to I/R
hearts exposed to filtered air [2]. Collectively, the results
suggest that polyamines play a role as possible cardiopro-
tectants to myocardium damage due to chronic ozone ex-
posure [5], which was previously unknown. Polyamines
(such as spermidine, cadaverine and putrescine) are linear
polycations with one or more amine groups and are in-
volved in cell synthesis of DNA and protein.
*This work was supported by grants from United States Environmental
Protection Agency Grant (USEPA Grant # IT-83404401-0), Texas A &
M Health Science Research Development Grant (Act# 134403-35402)
and funds from TAMHSC Research Startup (Act# 13100-35488),
Robert A. Welch Foundation (Departmental Grant AC006) and Na-
tional Science Foundation (NSF) Division of Undergraduate Education
(DUE) Program (9987332) for financial support. Dedicated to Karen
and Kevin Taylor.
†Dedicated to Karen and Kevin Taylor.
These metabolites are catalyzed by catabolic enzymes
such as ornithine decarboxylase and spermidine-N1-ace-
tyltransferase which aid synthesis and breakdown re-
spectively. The former also converts ornithine to putre-
scine and then converts spermidine and spermine in ac-
etylated form, which are converted to putrescine via po-

833 R. SETHI ET AL.
lyamine oxidase producing hydrogen peroxide and ami-
nopropionaldehyde in the process [6].
The above metabolite processes could under specific
circumstances contribute to cell stress/injury or I/R in-
jury. Furthermore polyamines have been linked to in-
creased susceptibility to hypertrophy; including a posi-
tive correlation between intracellular concentration of
spermine and calcium attenuation in isolated ventricular
myocytes from rats [7]. However, little is known regard-
ing polyamine metabolism and function in myocardial
I/R injury, particularly during oxidative stress.
Whereas, the oxidant nitric oxide (NO) is known to
react with superoxide and generate peroxynitrite anion,
also a strong oxidant, the effect of ozone is less known.
In addition, it has been documented that TNF alpha in-
duction of NO led to lowering of ODC activity including
inhibition of uptake of intracellular polyamines [8]. The
degree to which polyamine metabolism is influenced by
ozone in I/R hearts is unclear. This relationship is the
subject of this study with respect to concomitant altera-
tions of cellular polyamines in isolated rat hearts upon
O3-induced stress. The contributions of the work remain
the same as in the previous study, namely to elucidate the
“mechanism of action that results in a decrease in toler-
ance to myocardial ischemia” [2], however, unlike the
previous study focus on utilization of cadaverine as a
potential biomarker for oxidative stress. From the pro-
ceeding discussion it has been established that the main
polyamines involved in cell growth, differentiation and
apoptosis are putrescine, spermidine and spermine and
that the first rate-limiting enzyme in polyamine biosyn-
thesis is ornithine decarboxylase. It may seem instructive
to examine these enzymes and polyamines, however in
the current study this was not the case. In our study
therefore the focus was measurement of changes in the
other polyamines such as cadaverine, putrescine, and
spermidine, from which we observed an increase of sper-
midine in the experimental-to-control ratio; tentatively
indicate that a number of triggers are necessary for the
appropriate response.
2. Materials and Methods
Unless otherwise stated, all chemicals were reagent gra-
de, with ultrapure water. The workflow is summarized in
Figure 1.
2.1. Materials and Animals Used
All chemicals unless otherwise specified where obtained
from either Sigma-Aldrich (St. Louis, MO) or VWR In-
ternational (Chester, PA). The animals Sprague Dawley
rats (50 - 75 gm) were used under conditions previously
described [2].
2.2. Ozone Exposure Conditions
The procedure used was the same as previously descri-
bed [2]. Briefly, rats were kept within an environmental
chamber supplied with a constant air flow and subjected
to ozone as described previously [9].
2.3. Extraction Procedure
The procedure used was the same as previously descri-
bed [10]. Briefly, the frozen heart tissues were weighted
and homogenized in isotonic buffer at pH 7 and poly-
amines extracted as previously described [19].
2.4. Benzoylation Procedure
The polyamines were isolated from the homogenized
heart tissue through centrifugation and were derivatized
as previously described [11]. Benzoylated polyamines
were stored at –20˚C in all these previous methods.
2.5. HPLC Method
The method used was the same as previously described
[11]. Here, the solvent system was methanol (solvent A)
and water (solvent B) with a flow rate 1.0 ml/min (method
I) was throughout the isocratic method and the chromato-
graph for the experimental group is shown in Figure 2.
The benzoylated amines were extracted with diethyl
ether, which were eluted at room temperature through a
4.6 × 250 mm, 5 µm particle size reverse-phase C18 col-
umn which is detected at 254 nm. The time required for
completing one single run was 20 minutes and is shown in
Figure 3.
Figure 1. Workflow used in the experimental phase.

R. SETHI ET AL.
834
Figure 2. HPLC Chromatogram for procedure I for sepa-
ration of polyamines (40 minutes run).
Figure 3. HPLC Chromatogram for procedure II (same as I
except chloroform was used in the extraction step instead of
diethyl ether) for separation of polyamines (20 minutes run).
An alternative method described by Schotten-Bauma-
nn benzoylation procedure (procedure III, cited in [11])
was also used as a comparison to our chromatographic
procedure (Figure 4).
2.6. Statistical Analysis
All the data are presented as mean ± standard error (S.E.).
The biosynthesis and degradation processes of the dif-
ferent polyamines are connected, the relationship with
intracellular levels of these polyamines could be assessed
using either one-way or two-way ANOVA was used to
compare differences among groups where appropriate. A
two-way ANOVA could give us this information. To
differentiate between ozone-exposure hearts compared
with the control hearts Student’s t-test were performed.
Statistical comparison was performed by paired or un-
paired Students t-test. Significance level was setup at P <
0.05. The linear regression analysis was used to deter-
mine the correlation between different variables. In some
cases, ad hoc test was used.
3. Results and Discussion
Figures 2-3 show chromatograms for the standard po-
lyamines extracted from the control group. All chroma-
tograms (Figures 2-7) demonstrate baseline separation
and quantification of each polyamine cross-referenced to
authentic standards.
The chromatographs from experimental group were
compared with standards to identify specific polyamines
(Figures 5-6 of chromatograms corresponding to ex-
perimental group) which were identified and quantified.
The three polyamines were normalized for per gram of
fresh-weight heart tissue and compared and contrasted
with the control group. In addition, a statistical test was
undertaken to determine whether these differences were
statistically significant.
The comparison is shown in Figure 7 and for cada-
verine and spermidine the differences between the ex-
perimental and control group were significant at the 95%
level (P < 0.05) except for putrescine, due to biological
variation. To assess whether this variation was higher
than expected, a literature survey of standard error means
for other similar systems was examined and summarized
in Table 1.
Table 1 summarizes SE (Standard Error) values from
literature (research) in comparison to values from present
research. The significance of the results is discussed be-
low. The putrescine samples were diluted to allow accu-
rate measurements of cadaverine and spermidine.
From the analysis it appears that the biological varia-
tion in our study is comparable to other studies and not
an experimental artifact.
The results in Table 1 indicate the biological fluctua-
tions in polyamine content are within the general error
envelope indicated in other studies. The results also in-
dicate that during oxidative damage/stress, for example
ischemia, activities of the metabolite enzymes presuma-
bly ornithine decarboxylase spermidine/spermine N1–a-
cetyltransferase, polyamine oxidase are altered due to
Figure 4. HPLC Chromatogram for procedure III for
separation of polyamines 20 minutes run (same was I except
with heated column).

835 R. SETHI ET AL.
Figure 5. HPLC Chromatogram of experimental group with polyamines extracted and diluted with buffer at 1:8 ratio of
polyamine:buffer (w ith putrescine dilution, procedure I).
Figure 6. HPLC Chromatogram control group e xtracted and diluted with buffer at 1:8 ratio of polyamine:buffer (with pu-
trescine dilution, procedure I).
Figure 7. Plot of polyamine pool isolated from rat heart tissue corresponding to control group (white) and experimental
group (grey) with ANOVA statistical analysis at 95% significance (*) indicating that alterations of indigenous pool was statis-
tically significant for cadaverine and spermidine, of which the latter may confer some protection against I/R injury.
Table 1. Percentage of SE in polyamine values from previous researches and present research values.
% of SE in Cadaverine% of SE in Putrescine % of SE in Spermidine Others Works Reference
Control Test Control Test Control Test Control Test
28.12 23.43 18.56 27.84 12.51 21.92 ND ND Our Results
ND ND 31.201 32.521 18.371 15.282 11.991 22.303 19
ND ND 35.89 65.18 17.77 17.83 34.943 24.683 20
ND ND 14.28 30.4 26.09 36.07 203 253 21
ND ND 14.62 22.63 14.12 17.83 12.263 19.483 22
ND ND 28 59.25 8.06 44.77 15.813 38.223 23

R. SETHI ET AL.
836
induction triggered by ozone (O3) induced toxicity as
reflected in the measured concentrations. Putrescine levels
were increased, but were not statistically significant;
however, statistically significant were cadaverine levels
which decreased and spermidine levels which increased.
It is known the polyamine homoeostasis is dependent
upon ornithine decarboxylase, spermidine/spermine N1
-acetyltransferase and to a lesser degree lysine decar-
boxylase, depending upon the up-regulation of ornithine
decarboxylase, spermidine/spermine N1-acetyl-transfer-
ase, whereby changes in enzyme activity would in turn af-
fect putrescine content. We speculate putrescine levels to
decrease because if spermine N1-acetyltransferase were
down-regulated instead, then the expected increase in
putrescine would be marginal as observed in our study,
in other words any increase in the synthesis of cadaver-
ine by ornithine decarboxylase is offset by conversion to
spermidine by spermidine synthase and polyamine oxi-
dase, leading to an expected increase of spermidine
which was also observed. The magnitude of the poly-
amine would depend on the duration and strength of the
external stimuli [12]. The polyamine metabolism chan-
ges as observed in the heart in the present study might be
similar yet distinct to what has been observed in poly-
amine alterations in cerebral ischemia of polyamine stre-
ss response in brain [13]. These biological alterations
may be implicated in ischemia-related cell injury in heart
tissue. Free radical oxidant production and calcium ion
loading are two mechanisms implicated in the develop-
ment of myocardial I/R injury. As a result of O3 induced
injury spermidine levels increased to a greater exten than
putrescine levels which either increased slightly or re-
mained essentially constant indicating that polyamine
synthesis pathway was down-regulated and polyamine
degradation pathway was up-regulated. Ornithine decar-
boxylase is a rate-limited enzyme in the synthesis of pu-
trescine from ornithine and is susceptible to direct oxida-
tion, [14] leading to inactivation.
Similarly, spermidine/spermine N1-acetyltransferase
catalyzes the acetylation of putrescine to spermidine,
which upon induction by various NO or toxins or stress,
in conjugation with polyamine oxidase could lead to a
decrease of spermine and production of hydrogen perox-
ide and aldehyde by-product.
Up-regulation may decrease spermine mitigating oxi-
dative stress response. However, a decrease in spermine
levels may not correspond to a similar decrease in sper-
midine levels, since the latter can also be synthesized by
spermidine synthase affecting other biological systems
regulated by polyamines. Systems regulated by poly-
amines include free radical scavenging and reduction in
lipid peroxidation [15].
Although speculative, up-regulation of spermidine/
spermine N1-acetyltransferase could lead to the genera-
tion of hydrogen peroxide and aldehydes in ischemic
hearts and alterations to spermine and possible sper-
midine (↑) and calcium ions (↑), ameliorating any posi-
tive changes in protection, [16] through DNA base-pair
stabilization from oxidative stress, or protection from
endonucleases.
This is because there is a body of literature which con-
nects I/R injury to alterations in spermine [17] and stress
in a number of animal/plant models including use of di-
fluoromethylornithine (DFMO) as a potent inhibitor of
polyamine biosynthesis, Ornithine decarboxylase (ODC)
[18], therefore I/R injury and alterations in ROS and/or
NO levels and quantification of apoptosis in the different
groups has been established as a leading hypothesis in
the mechanism of injury via spermine/ODC alterations.
Once the mechanism is trigged an increases in sper-
midine levels can provid a cardioprotective effect. This
protection from oxidative damage from O3-induced stre-
ss may be through cell membrane stabilization and in-
creased scavenging of oxygen radical species in addition
to preventing calcium ion loading [18].
Consistent with earlier observations from our previous
study that both malondialdehyde and TNF alpha levels
increased with decreases in interlukin-10 (IL-10) levels
is indicative of increased myocardial oxidative stress
levels [2].
O3-mediated lipid peroxidation is counted by radical
scavengers, such as SOD or spermidine through binding
to membrane phospholipids or formation of a polycation-
complex through modification of the auto oxidation of
Fe2+ [19] leading to inhibition of lipoxygenase, there- by
minimizing peroxidative damage. Since SOD is a key
enzyme for detoxification of superoxide anions, gener-
ated by oxidants such as O3, lowering of SOD/glutathi-
one reductase and glutathione S-transferase glutathione
activity (free glutathione) may be countered by polyami-
nes such as spermidine/spermine, which has been de-
monstrated in plants [20] including O3-induced leaf ne-
crosis in tobacco [21]. Our model consistent with our
previous findings and current study indicates that upon
generation ozone TNF alpha is induced leading to genera-
tion of reactive oxygen species (ROS) induced apoptosis
through activation of JNK [22]. In response to this stress,
the cell utilizes anti-oxidants such as glutathione, or anti-
oxidant enzymes such as SOD. Concurrent to SOD ex-
pression, polyamines, particularly spermidine/spermine
reduce TNF alpha-induced apoptosis. It has also been
shown that JNK and p38 proteins (proapoptotic mediators
from the MAPK family of proteins) are activated during
UV/oxidative stress [23]. From the above outline, it can
be summarized that JNK activation, either directly

837 R. SETHI ET AL.
or indirectly via proapoptotic mediator by TNF alpha
would lead to apoptosis. Inhibition I/R-induced oxidative
stress of TNA alpha by IL-10 as a first response (early
phase), plus effect of glutathione, SOD (early/intermedi-
ate phase) and alterations in polyamine content (e.g. de-
pletion of cadaverine, increase in intermediate/late phase)
confer a limited degree of protection. The effect of poly-
amines on JNK inactivation through increased ERK (an-
tiapoptotic mediators from the MAPK family of proteins)
through dephosphorylation. The protective effects of
ERK in cardiomyocytes have already been documented
[24] through the Bcl-2 family of proteins [25] with re-
lease of cytochrome c, although in our study this was not
investigated.
4. Conclusions
In summary, prolonged exposure to ozone gave rise to
increased putrescine (approximately 1.06-fold relative to
control) and spermidine (1.57-fold) concentration, and
decreased cadaverine (4.54-fold) concentration. From the
above results spermidine levels may be compensating the
changes in putrescine and cadaverine, but only up to cer-
tain levels. The results were pair-wise compared and
were found to be significant for cadaverine and spermi-
dine with biological variation within other published lite-
rature values.
These results indicate that O3 can activate myocardial
polyamine stress response pathway(s) resulting in en-
hanced I/R injury. This injury can be attributed to altera-
tions in cadaverine and putrescine concentrations in the
myocardium. We believe that the increase in spermidine
levels seen in our study may be involving a compensatory
response to O3 induced ischemic injury. The mechanism
of depletion may activate certain pathways, such as inhi-
bition of TNF alpha, which in turn prevents JNK activa-
tion and promotes apoptosis via MAPK family of proteins.
These proteins regulate apoptotic signaling via ERK ac-
tivation, which has been observed in other studies.
Lastly, we believe that indigenous spermidine stabi-
lizes the intracellular polyamine pool which in turn pro-
vides limited protection against I/R injury.
5. Acknowledgements
We are grateful to Mohammad T. Nutan (TAMHSC) for
giving permission to use the HPLC and Mr. Don Marek
from the Department of Environmental Engineering
(TAMUK) for assistance with the glove box.
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839 R. SETHI ET AL.
Author Contribution
S. R. Chava undertook all of the experimental work (ex-
traction, triplicate analysis) related to the chromatography.
S. Bashir assisted in the extraction and the first set of
analyses. He wrote the first and co-wrote the second and
submission draft of the manuscript. M. Castro and R. Sethi
co-supervised the student towards his Master of Science
thesis. R. Sethi undertook the entire animal component
and wrote the first paper (Ref [2]) and conferred on the
current submitted draft. Finally, Dr. Castro assisted with
the publication costs.
Abbreviations
ANOVA Analysis Of Variance
Bcl-2 B cell leukaemia-2
CD20 Cluster of differentiation twenty
Cad Cadaverine
DFMO Difluoromethylornithin
CVD Cardiovascular disease
DNA Deoxyribonucleic acid
ERK Extracellular signal-regulated
kinase pathway protein
Fe2+ Iron (II) cation
HPLC High performance liquid
chromatography
I/R Ischemia reperfusion
IL-10 Interleukin-10
JNK c-Jun NH2-terminal protein kinases
LVEDP Left ventricular end diastolic pressure
MALT Mucosa associated lymphoid tissue
MAPK Mitogen activated protein kinase
ND Not determined
NO Nitric oxide
O3 Ozone
ODC Ornithine decarboxylase
P-value Probability value (estimation of
Randomness)
Put Putrescine
ROS Reactive oxygen species
SOD Superoxide dismutase
Student t-testTesting the difference between means
TNF alpha Tumor necrosis factor-alpha
U
V Ultraviolet