Role of Polyamines in Ozone Exposed Ischemic-Reperfused Hearts

doi:10.4236/ajac.2011.27095

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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

E-mail: #[email protected], #kfmec00@tamuk.edu

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.

6. References

[1] T. Thom, N. Haase, W. Rosamond, V. J. Howard, J. Ru-

msfeld, T. Manolio, Z. J. Zheng, K. Flegal, C. O’Donnell,

S. Kittner, D. Lloyd-Jones, D. C. Goff, Y. Hong, R. Ad-

ams, G. Friday, K. Furie, P. Gorelick, B. Kissela, J. Mar-

ler, J. Meigs, V. Roger, S. Sidney, P. Sorlie, J. Steinber-

ger, S. Wasserthiel-Smoller, M. Wilson and P. Wolf,

“Heart Disease and Stroke Statistics-2006 Update: A Re-

port from the American Heart Association Statistics

Committee and Stroke Statistics Subcommittee,” Circu-

lation, Vol. 113, 2006, pp. E85-E151.

doi:10.1161/CIRCULATIONAHA.105.171600

[2] R. S. P. Perepu, C. Garcia, D. Dostal and R. Sethi, “En-

hanced Death Signaling in Ozone Exposed Ischemic-

Reperfused Hearts,” Molecular and Cellular Biochemis-

try, Vol. 336, No. 1-2, 2010, pp. 55-64.

doi:10.1007/s11010-009-0265-4

[3] K. K. Sathish, M. Haque, T. E. Perumal, J. Francis and R.

M. Uppu, “A Major Ozonation Product of Cholesterol, 3-

Beta-Hydroxy-5-Oxo-5, 6-Seco-Cholestan-6-al, Induces

Apoptosis in H9c2 Cardiomyoblasts,” FEBS Letters, Vol.

579, No. 28, 2005, pp. 6444-6450.

doi:10.1016/j.febslet.2005.10.044

[4] R. D. Brooke, “Cardiovascular Effects of Air Pollution,”

Clinical Science, Vol. 115, 2008, pp. 175-187.

doi:10.1042/CS20070444

[5] J. B. Ruidavets, M. Cournot, S. Cassadou, M. Giroux, M.

Meybeck and J. Ferrieres, “Ozone Air Pollution Is Asso-

ciated with Acute Myocardial Infarction,” Circulation,

Vol. 111, 2005, pp. 563-569.

doi:10.1161/01.CIR.0000154546.32135.6E

[6] K. Niiranen, M. Pietilä, T. J. Pirttilä, A. Järvinen, M.

Halmekytö, V. P. Korhonen, T. A. Keinänen, L. Alhonen

and J. Jänne, “Targeted Disruption of Spermidine/Sper-

mine N1-Acetyltransferase Gene in Mouse Embryonic

Stem Cells. Effects on Polyamine Homeostasis and Sen-

sitivity to Polyamine Analogues,” Journal of Biological

Chemistry, Vol. 277, 2002, pp. 25323-25328.

doi:10.1074/jbc.M203599200

[7] R. Wang, C. Q. Xu, W. M. Zhao, J. Zhang, K. Cao, B. F.

Yang and L. F. Wu, “Calcium and PolyaminE Regulated

Calcium-Sensing Receptors in Cardiac Tissues,” Euro-

pean Journal of Biochemistry, Vol. 270, No. 12, 2003, pp.

2680-2688. doi:10.1046/j.1432-1033.2003.03645.x

[8] J. Satriano, S. Ishizuka, D. C. Archer, R. C. Blantz and C.

J. Kelly, “Regulation of Intracellular Polyamine Biosyn-

thesis and Transport by NO and Cytokines TNF-Alpha

and IFN-Gamma,” American Journal of Physiology, Vol.

276 (4 Pt 1), 1999, pp. C892-C899.

[9] M. J. Wiester, J. S. Tepper, M. E. King, M. G. Menache

and D. L. Costa, “Comparative Study of Ozone (O3) Up-

take in Three Strains of Rats and in the Guinea Pig,”

Toxicology and Applied Pharmacology, Vol. 96, No. 1,

1998, pp. 140-146. doi:10.1016/0041-008X(88)90256-6

[10] R. Sethi, N. S. Dhalla, “Inotropic Responses to Isopro-

terenol in Congestive Heart Failure Subsequent to Myo-

cardial Infarction in Rats,” Journal of Cardiac Failure,

Vol. 1, No. 5, 1995, pp. 391-399.

doi:10.1016/S1071-9164(05)80008-9

R. SETHI ET AL.

838

[11] R. Sethi, S. R. Chava, S. Bashir and M. E. Castro, “An

Improved High Performance Liquid Chromatographic

Method for Identification and Quantization of Poly-

amines as Benzoylated Derivatives,” American Journal of

Analytical Chemistry, Vol. 2, 2011, pp. 456-469.

doi:10.4236/ajac.2011.24055

[12] Y. Hayashi, J. Tanaka, Y. Morizumi, Y. Kitamura and Y.

Hattori, “Polyamine Levels in Brain and Plasma after

Acute Restraint or Water-Immersion Restraint Stress in

Mice,” Neuroscience Letters, Vol. 355, No. 1-2, 2004, pp.

57-60. doi:10.1016/j.neulet.2003.10.027

[13] B. A. Coert, R. E. Andereson and F. B. Meyer, “Exoge-

nous Spermine Reducesischemic Damage in a Model of

Focal Cerebral Ischemia in the Rat,” Neuroscience Let-

ters, Vol. 282, No. 1-2, 2000, pp. 5-8.

doi:10.1016/S0304-3940(00)00856-9

[14] J. M. Dypbukt, M. Ankarcrona, M. Burkitt, A. Sjoholm,

K. Strom, S. Orrenius and P. Nicotera, “Different Pro-

oxidant Levels Stimulate Growth, Trigger Apoptosis, or

Produce Necrosis of Insulin-Secreting RINm5F Cells,”

Journal of Biological Chemistry, Vol. 269, 1994, pp.

30553-50560.

[15] K. C. Das and H. P. Misra, “Hydroxyl Radical Scaveng-

ing and Singlet Oxygen Quenching Properties of Poly-

amines,” Molecular and Cellular Biochemistry, Vol. 262,

No. 1-2, 2004, pp. 127-133.

doi:10.1023/B:MCBI.0000038227.91813.79

[16] J. M. Ribeiro and D. A. Carson, “Ca2+/Mg2+-Dependent

Endonuclease from Human Spleen: Purification, Proper-

ties and Role in Apoptosis,” Biochemistry, Vol. 32, No.

35, 1993, pp. 9129-9136. doi:10.1021/bi00086a018

[17] G. Drolet, E. B. Dumbroff, R. L. Legge and J. E. Thom-

pson, “Radical Scavenging Properties of Polyamines,”

Phytochemistry, Vol. 25, No. 2, 1986, pp. 367-371.

doi:10.1016/S0031-9422(00)85482-5

[18] R Alcázar, T. Altabella, F. Marco, C. Bortolotti, M.

Reymond,·C. Koncz, P. Carrasco and A. F. Tiburcio,

“Polyamines: Molecules with Regulatory Functions in

Plant Abiotic Stress Tolerance,” Planta, Vol. 231, 2008,

pp. 1237-1249.

[19] G. Noctor, A. M. Arisi, L. Jouanin, K. J. Kunert, H. Ren-

nenberg and C. Foyer, “Glutathione: Biosynthesis, Me-

tabolism and Relationship to Stress Tolerance Explored

in Transformed Plants,” Journal of Experimental Botany,

Vol. 49, 1988, pp. 623-647.

doi:10.1093/jexbot/49.321.623

[20] C. Langebartels, K. Kerner, S. Leonardi, M. Schraudner,

M. Trost, W. Heller and H. Sandermann, “Jr. Biochemi-

cal Plant Responses to Ozone. Differential Induction of

Polyamine and Ethylene Biosynthesis in Tobacco,” Plant

Physiology, Vol. 95, 1991, pp. 882-889.

doi:10.1104/pp.95.3.882

[21] Z. G. Liu, H. Hsu, D. V. Goeddel and M. Karin, “Dissec-

tion of TNF Receptor 1 Effector Functions: JNK Activa-

tion Is Not Linked to Apoptosis While NF-kB Activation

Prevents Cell Death,” Cell, Vol. 87, No. 3, 1996, pp. 565-

576. doi:10.1016/S0092-8674(00)81375-6

[22] J. M. Kyriakis and J. Avruch, “Protein Kinase Cascades

Activated by Stress and Inflammatory Cytokines,” Bio-

essays, Vol. 18, No. 7, 1996, pp. 567-577.

doi:10.1002/bies.950180708

[23] R. Aikawa, I. Komuro, T. Yamazaki, Y. Zou, S. Kudoh,

M. Tanaka, I. Shiojima, Y. Hiroi and Y. Yazaki, “Oxida-

tive Stress Activates Extracellular Signal Related Kinases

through Src and Ras in Cultured Cardiac Myocytes of

Neonatal Rats,” Journal of Clinical Investigation, Vol.

100, No. 7, 1997, pp. 1813-1821. doi:10.1172/JCI119709

[24] M. Mori, M. Uchida, T. Watanabe, K. Kirito, K. Hatake,

K. Ozawa and N. Komatsu, “Activation of Extracellular

Signal Related Kinases ERK1 and ERK2 Induces Bcl-xL

up-Regulation via Inhibition of Caspase Activities in Ery-

thropoietin Signaling,” Journal of Cell Physiology, Vol.

195, No. 2, 2003, pp. 290-297. doi:10.1002/jcp.10245

[25] T. E. Soderstrom, M. Poukkula, T. H. Holmstrom, K. M.

Heiskanen and J. E. Eriksson, “Mitogen-Activated Pro-

tein kinase/Extracellular Signal-Related Kinase Signaling

in Activated T Cells Abrogates TRAIL-Induced Apop-

tosis Upstream of the Mitochondrial Amplification Loop

and Caspase-8,” Journal of Immunology, Vol. 169, 2002,

pp. 2851-2860.

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

V Ultraviolet