Inhibition of peroxynitrite-mediated cellular toxicity, tyrosine nitration,and a1-antiproteinase inactivation by 3-mercapto-2-methylpentan-1-ol,

a novel compound isolated from Allium cepaq

Peter Rose,a Sabine Widder,b Jan Looft,b Wilhelm Pickenhagen,c

Choon-Nam Ong,a and Matthew Whitemanc,*

a Department of Community, Occupational and Family Medicine, Faculty of Medicine, National University of Singapore, 8 Medical Drive, Singaporeb Corporate research division, DRAGOCO Gerberding AG, D-37603, Holzminden, Germany

c Department of Biochemistry, Faculty of Medicine, National University of Singapore, 8 Medical Drive, Singapore

Received 20 January 2003

Abstract

Peroxynitrite formation in vivo is implicated in numerous human diseases and there is considerable interest in the use of antiox-

idants and natural products such as thiols as ‘‘peroxynitrite scavengers’’. We therefore investigated the effects of a recently identified

constituent of onions, 3-mercapto-2-methylpentan-1-ol (3-MP), for its ability to inhibit peroxynitrite-mediated processes in vitro and

using cultured human cells and compared its effectiveness against glutathione. 3-MP significantly inhibited peroxynitrite-mediated

tyrosine nitration and inactivation of a1-antiproteinase to a greater extent than glutathione at each concentration tested (15–500 lM).

3-MP also inhibited peroxynitrite-induced cytotoxicity, intracellular tyrosine nitration, and intracellular reactive oxygen species

generation in human HepG2 cells in culture to a greater extent than glutathione. These data suggest that 3-MP has the potential to act

as an inhibitor of ONOO�-mediated processes in vivo and that the antioxidant action of 3-MP deserves further study.

� 2003 Elsevier Science (USA). All rights reserved.

Keywords: 3-Nitrotyrosine; 3-Mercapto-2-methylpentan-1-ol; Peroxynitrite; Allium; Antioxidants; a1-Antiproteinase

The interaction of nitrogen monoxide (�NO) and su-

peroxide (O��2 ) forms the cytotoxic product peroxyni-

trite, (ONOO�) (Eq. (1)) [1].�NOþO2

�� ! ONOO� ð1ÞThe rate constant for this reaction is greater than

109M�1 s�1 [2]. Under physiological conditions ONOO�

has a half life of under 1 s and is converted to its pro-

tonated form peroxynitrous acid, ONOOH, which in

turn decays to generate multiple toxic products with

reactivities resembling those of the nitryl cation (NOþ2 ),

nitrogen dioxide radical (�NO2), and hydroxyl radical

(�OH). Peroxynitrite and species derived from it canoxidise lipids, proteins, DNA, and carbohydrates [3–5].

The addition of ONOO� to biological fluids leads to the

depletion of ascorbate, urate, and thiols [6]. Cells ex-

posed to ONOO� show oxidative DNA damage, DNA

strand breakages [7], and activation of poly(ADP-ribo-

syl)polymerase [8] and caspase-3 [9] as well as complex

intracellular signalling involving the activation of sev-

eral MAP Kinases including ERK1/2, p38, JNK, andSrc [10–13]. Furthermore, ONOO� reacts with the

amino acid tyrosine to form a nitrated bio-marker, 3-

nitrotyrosine (reviewed in [14,15]). Increased levels of

this nitrated amino acid have been detected in numerous

diseases and animal models of pathological conditions

Biochemical and Biophysical Research Communications 302 (2003) 397–402

www.elsevier.com/locate/ybbrc

BBRC

qAbbreviations: a1-AP, a1-antiproteinase; DCF-DA, dichlorofluo-

rescein diacetate; DMSO, dimethyl sulphoxide; DNA, deoxyribonu-

cleic acid; EBSS, Earles balanced salt solution; K2HPO4, dipotassium

hydrogen phosphate; DTNB 5,50-dithio-bis(2-nitrobenzoic acid); GSH,

reduced glutathione; KH2PO4, potassium dihydrogen phosphate; LDL,

low density lipoprotein; MEM, minimal essential media; 3-MP,

3-mercapto-2-methylpentan-1-ol; MTT, 3-(4,5-dimethyl-2-yl)-2,5-

diphenyltetrazolium bromide; ONOO�, peroxynitrite; NO2Cl, nitryl

chloride; �NO, nitric oxide; PBS, phosphate-buffered saline; RNS,

reactive nitrogen species; SANA, N-succinyl ðalaÞ3 p-nitroanilide.* Corresponding author. Fax: +65-6779-1453.

E-mail address: bchwml@nus.edu.sg (M. Whiteman).

0006-291X/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.

doi:10.1016/S0006-291X(03)00193-1

including atherosclerosis, coronary artery disease, gas-trointestinal cancers, cirrhosis, and hepatitis infection

(reviewed in [14,15]).

Due to the cytotoxicity of ONOO� and its apparent

formation at sites of tissue injury [14,15], there has been

considerable interest in the ability of natural and syn-

thetic antioxidants to interfere with ONOO�-mediated

damage [16–18]. Plant derived antioxidants such as

flavonoids have been proposed to fulfill this role [19,20]and much research has focused on the potential anti-

oxidant and cytoprotective or anti-carcinogenic prop-

erties of flavonoids and related compounds [21–25].

Allium species are a major source of flavonoids in the

human diet [26], in addition they also contain large

quantities of organosulphur compounds such as S-Al-

k(en)yl-LL-cysteine sulphoxides, and thiolsulphinates,

many of which also show potent antioxidant and anti-carcinogenic properties in vitro [23,27–29]. Recently, an

additional ‘‘novel’’ organosulphur compound, 3-mer-

capto-2-methylpentan-1-ol (3-MP), was identified in

Allium cepa that is formed during tissue damage [30,31].

However, there has been no research to determine

whether 3-MP has any potential antioxidant or cyto-

protective properties. This is of scientific interest as ep-

idemiological studies have shown an inverse correlationbetween long-term risk of cardiovascular disease and

gastric cancers with consumption of Allium vegetables,

in addition to them having potent anti-bacterial, anti-

thrombic, and lipid lowering attributes [21–23].

In the present paper, the ability of 3-MP to prevent

ONOO�-mediated damage was examined using model

systems; inhibition of cell toxicity, inhibition of intra-

cellular and in vitro tyrosine nitration, and inhibition ofa1-antiproteinase inactivation. Furthermore, its effec-

tiveness was compared to glutathione. Intracellular ty-

rosine nitration is observed in vivo [14,15] and the

addition of ONOO� to cultured cells causes substantial

cytotoxicity and cell death by apoptotic mechanisms

[7–9]. a1-AP inactivation is also observed in vivo [32,33]

at sites of inflammation and ONOO� formation pro-

vides a plausible mechanism [34]. Hence it was logical touse these target molecules in assays of agents able to

protect against ONOO�-dependent damage.

Materials and methods

Materials. Hydrogen peroxide (30% solution), manganese dioxide,

and HPLC grade methanol were purchased from BDH (Poole, Dorset,

England). Tyrosine, a1-antiproteinase (A9024), porcine elastase

(E0258), and N-succinyl ðalaÞ3-p-nitroanilide (SANA), and all other

reagents were obtained from Sigma–Aldrich (St. Louis, MO, USA).

Distilled water passed through a Millipore water purification system

was used for all purposes. Synthesis of 3-mercapto-2-methylpentan-1-

ol and hydrogen peroxide-free peroxynitrite (ONOO�) was essentially

as described [31] and [1], respectively. ONOO� solution was quantified

immediately before use using a molar absorption coefficient of

1670cm�1M�1 [35].

Cell culture. The human hepatoma HepG2 cell line was purchased

from the American Type Culture Collection (Rockville, MD, USA),

cultured in minimum essential media (MEM) supplemented with 1%

penicillin/streptomycin, 10% foetal bovine serum, and grown at an

atmosphere containing 5% CO2:95% O2 with �95% humidity to 90%

confluency before use.

Determination of partition coefficient. The determination of parti-

tion coefficients, a measure of the liphophilic nature of a compound,

was essentially the same as described in [36,37]. In brief, 1mM of each

compound investigated was added to an n-octanol/water (1:1) mix and

vortexed for 5min at room temperature. Samples were then left at

room temperature for 30min before an aliquot of either the n-octanol

or water fraction was used to determine the concentration of each thiol

compound using the DTNB method described by Tietz and Griffiths

[38,39]. A solution of n-octanol was used as a blank.

The effect of 3-MP on ONOO�-mediated cell toxicity; comparison

with GSH. Cellular metabolic activity was estimated using 3-(4,5-di-

methyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as described [7].

Cells were seeded overnight at a density of 0:25� 106 cells/well in 24-

well microplates and washed twice with warm (37 �C) PBS before

addition of 3-MP or GSH for 10min. ONOO� (500lM) was added for

5min. Cells were then washed twice with warm (37 �C) PBS and 200llof warm (37 �C) serum-free MEM containing MTT (0.5mg/ml) was

added for 1 h. MTT reduction was measured using a Molecular De-

vices Spectramax190 plate reader (Sunnyvale, Ca, USA) after solu-

bilisation of the formazan dye with 200ll DMSO and gentle shaking in

the dark for 30min.

To estimate intracellular reactive oxygen species generation, cells

were loaded in foetal calf serum-free MEM containing 10lM dichlo-

rofluorescein diacetate for 1 h at 37 �C, as described [40]. Cells were

then gently washed with warm PBS (37 �C) before addition of 3-MP or

GSH for 10min. After this time, ONOO� was added and increases in

fluorescence were measured using a Molecular Devices GeminiXS

fluorescence microplate reader with excitation and emission wave-

lengths set at 490 and 535nm, respectively.

Prevention of a1-antiproteinase inactivation. Elastase and a1-anti-

proteinase (a1-AP) were measured essentially as described in [16].

Briefly, 40 ll of a1-AP stock solution (4mg/ml) was incubated in

100mMK2HPO4–KH2PO4 buffer, pH 7.4, and various antioxidants in

a water bath for 15min at 37 �C. After this time, ONOO� (500lM)

was added, the samples were incubated for 5min, and 2.0ml phosphate

buffer was added. Residual elastase activity was measured by the ad-

dition of 100ll elastase substrate (succinyl ðAlaÞ3 p-nitroanilide,

SANA; 4mg/ml) and the change in absorbance at 410 nm was mea-

sured for 30 s. To assess the role of ONOO� decomposition products

on a1-AP activity, ‘‘decomposed’’ ONOO� was prepared by adding

500lM ONOO� to 100mM phosphate buffer, pH 7.4, at 37 �C 5min

prior to the addition of a1-antiproteinase or antioxidants [16].

Measurement of tyrosine nitration; comparison with GSH. The ad-

dition of ONOO� to the amino acid tyrosine results in substantial ty-

rosine nitration and formation of 3-nitrotyrosine [16] and has been used

extensively to assess ONOO� scavenging activity [16,41,42]. Tyrosine

nitration was measured as described in [16]. Briefly, 1mM tyrosine was

incubated in a solution containing 100mM K2HPO4–KH2PO4 buffer,

pH 7.4, and various antioxidants in a water bath for 15min at 37 �C.After this time, 1mM ONOO� was added and the solutions were fur-

ther incubated for 15min. Reverse phase HPLC analysis was performed

using a Spherisorb 5ODS2 C18 column with a mobile phase consisting

of 100mM K2HPO4–KOH buffer, pH 3.01, with 20% (v/v) methanol.

Peak area (retention time of 3.8min) was measured and concentrations

were calculated from a standard curve. The detection limit for 3-NO2

tyrosine was 0.1lM.

To examine the extent to which 3-MP and GSH could inhibit in-

tracellular protein tyrosine nitration, human HepG2 cells were incu-

bated with various concentrations of 3-MP for 5min and sublethal

concentrations of ONOO� (100 lM) were added for 5min. After this

time, cells were washed three times and lysed with 10mMHepes buffer,

398 P. Rose et al. / Biochemical and Biophysical Research Communications 302 (2003) 397–402

pH 7.4, containing 50mM b-glycerophosphate, 70mM NaCl, 2mM

EDTA, and 1% SDS. Intracellular tyrosine nitration was measured

using a commercial ELISA kit (Chemicon, #HK501) following the

manufacturer�s instructions.Data analysis. All graphs are plotted with means standard devi-

ation of the mean (SD). In all cases the mean values were calculated

from data taken from at least six separate experiments performed on

separate days using freshly prepared reagents. Where significance

testing was performed, an independent t test (Student�s; 2 populations)

was used; *p < 0:05, **p < 0:01, and ***p < 0:001.

Results

Determination of oil:water partition coefficient

Using n-octanol/water, the partition coefficients

[36,37] for 3-MP and GSH were determined to be

10 0:1 and 0:01 0:001, respectively. Therefore, 3-MP

is 1000-fold more lipophilic than GSH.

Inhibition of tyrosine nitration

The addition of ONOO� to a solution of tyrosine ledto the formation of 3-nitrotyrosine [1]. As expected, the

addition of 1mM ONOO� to a buffered solution of

1mM tyrosine led to the formation of 70–80 lM of 3-

nitrotyrosine [16]. 3-MP significantly inhibited ONOO�-

mediated tyrosine nitration at all concentrations tested

Fig. 1. Inhibition of ONOO�-mediated tyrosine nitration by 3-MP:

comparison with GSH. Tyrosine (1mM) incubatedNaCO3 (25mM)

in phosphate buffer, pH 7.4, at 37 �C for 15min antioxidants and

ONOO� (1mM) added. 3-NO2 tyrosine formed was measured by

HPLC as described in Materials and methods. Data are means SD of

six or more independent experiments. Significance testing was per-

formed using an independent t test (Student�s; 2 populations); *p <

0:05, **p < 0:01, and ***p < 0:001 comparing 3-MP to GSH.

Fig. 2. Inhibition of ONOO�-mediated a1-AP inactivation by 3-MP: comparison with GSH. (A) ONOO� (500lM) was added to a1-AP in the

presence of increasing concentrations of 3-MP followed by the addition of elastase. Control experiments (B–D). No direct effects of 3-MP on elastase

or the ability of a1-AP to inhibit elastase (B), lack of protective effects of 3-MP or GSH on a1-AP inactivation following pre-treatment with ONOO�

(C) and pre-treatment of a1-AP with either 3-MP or GSH did not reverse ONOO�-mediated inactivation of a1-AP (D). Data are meansSD of six

or more independent experiments. Significance testing was performed using an independent t test (Student�s; 2 populations); *p < 0:05, **p < 0:01,

and ***p < 0:001 comparing 3-MP to GSH.

P. Rose et al. / Biochemical and Biophysical Research Communications 302 (2003) 397–402 399

(15–500 lM) (Fig. 1). Even at low concentrations(15 lM) compared to 1mM ONOO�, 3-MP significantly

decreased ONOO�-mediated 3-nitrotyrosine formation

to a greater extent than that of the positive control,

reduced glutathione, known to be a powerful scavenger

of ONOO� [1,16].

Inhibition of ONOO�-mediated inactivation of a1-AP

In agreement with previous studies [16] the additionof ONOO� led to substantial inactivation of a1-AP. A

concentration of 500 lM ONOO� was chosen from

preliminary experiments to achieve substantial but not

complete inactivation of a1-AP so that both decreases

and increases in the extent of inactivation could be ob-

served [41,42]. Fig. 2A shows that addition of 30 lM of

3-MP substantially protected a1-AP from ONOO�-

mediated inactivation and when tested in the concen-tration range 60–250 lM 3-MP offered significantly

more protection than equimolar concentrations of the

positive control, GSH (Fig. 2A). In contrast to certain

other thiol compounds such as penicillamine, cysteine,

or captopril [41,42], no aggravation of ONOO�-medi-

ated inactivation of a1-AP was observed at low

(<30 lM) concentrations of 3-MP.

Figs. 2B–D show control experiments. For example,3-MP had no direct effect on elastase or the ability of a1-

AP to inhibit elastase (Fig. 2B). If 3-MP or GSH were

added to the reaction mixture 5min after the addition of

ONOO� to a1-AP, it had no effect, neither stimulating

nor decreasing inactivation (Fig. 2C). Since inactivation

of a1-AP is complete within 5min [16], it follows that 3-

MP is not acting by altering the residual activity of a1-

AP after damage by ONOO�. Similarly, incubation of3-MP (or GSH) with ONOO� for 5min prior to addi-

tion of a1-AP did not give any observable inactivation of

a1-AP subsequently added (Fig. 2D). Thus, the products

of the reaction of 3-MP (and GSH) with ONOO� do not

persist in the reaction mixture or inactivate a1-AP.

Inhibition of ONOO�-induced cellular toxicity and intra-

cellular tyrosine nitration

Incubating HepG2 cells for 18 h in the presence of up

to 500 lM 3-MP or GSH did not result in any significant

cell toxicity measured using the MTT assay and resulted

in 2:3 1:8% and 3:5 2:2% losses of viability, respec-

tively, i.e., 3-MP itself was not cytotoxic. Whereas the

addition of 500 lM ONOO� led to substantial cyto-

toxicity, reducing cell viability to (22:9 6:9%). How-

ever, in the presence of 30 lM, 3-MP or 60 lM GSH,compared to 500 lM ONOO�, significant inhibition of

ONOO�-mediated loss of cell viability was observed

(Fig. 3A). Interestingly, at concentrations <125 lM and

P250 lM 3-MP offered significantly higher protection

than equimolar GSH.

We also investigated whether 3-MP could inhibit

ONOO�-mediated formation of intracellular reactive

oxygen species detected by DCF-DA. The addition of

500 lMONOO� to cells preloaded withDCF-DA caused

Fig. 3. Inhibition of ONOO�-mediated cytotoxicity to HepG2 cells:

comparison with GSH. Cells were treated with either 3-MP or GSH and

ONOO� added. Cell viability was assayed usingMTT (A), formation of

intracellular peroxides was assayed using dichlorofluorescein diacetate

(B), and intracellular 3-nitrotyrosine formation was assayed using a

commercial ELISA, as described in Materials and methods. Data are

means SD of six or more independent experiments. Significance test-

ingwas performed using an independent t test (Student�s; 2 populations);*p < 0:05, **p < 0:01, and ***p < 0:001 comparing 3-MP to GSH.

400 P. Rose et al. / Biochemical and Biophysical Research Communications 302 (2003) 397–402

a large increase in intracellular reactive oxygen speciesand associated DCF fluorescence. However, the addition

of ONOO� in the presence of 3-MP and to a lesser extent

GSH (Fig. 3B) significantly inhibited the formation of

ONOO�-mediated intracellular reactive oxygen species

(ROS) detected using DCF. At concentrations

P125 lM, 3-MP significantly inhibited ONOO�-medi-

ated intracellular ROS formation to a greater extent than

equimolar concentrations of GSH (Fig. 3B). Controlexperiments showed that incubating HepG2 cells with up

to 500 lM 3-MP or GSH for 1 h did not cause significant

formation of DCF-detectable ROS (data not shown), i.e.,

neither 3-MP or GSH caused intracellular ROS forma-

tion.

In addition, a frequently used bio-marker for RNS

formation in vivo is 3-nitrotyrosine formed from the

reaction of RNS with the amino acid tyrosine (reviewedin [14,15]). Therefore, we investigated whether 3-MP

could inhibit ONOO�-mediated intracellular tyrosine

nitration using a commercially available ELISA. The

addition of 100 lM ONOO� to HepG2 cells resulted in

the formation of 9 0:3 nmol/3-nitrotyrosine per milli-

gram protein. However, treating HepG2 cells with

P15 lM of either 3-MP or GSH for 5min prior to

ONOO� addition significantly inhibited ONOO�-medi-ated intracellular tyrosine nitration (Fig. 3C).

Discussion

The generation of peroxynitrite in vivo is implicated in

a wide range of human diseases ranging from cancer and

cardiovascular diseases to chronic inflammation and in-fluenza infection (reviewed in [14,15]). Both free and

protein bound 3-NO2 tyr are found in vivo. For example,

3-NO2 tyr in the free form is found in rheumatoid

arthritis, amyotrophic lateral sclerosis, and protein

bound 3-NO2 tyr is found in acute lung injury, athero-

sclerosis, gastrointestinal inflammatory cancers, amyo-

trophic lateral sclerosis, Parkinson�s disease, and

myocardial inflammation (reviewed in [14,15]). The for-mation of 3-NO2 tyr is thought to contribute to the

overall disease pathology by interfering with signal

transduction mechanisms involving phosphorylation/

dephosphorylation [43], adenylation [44], and protein

assembly [45] whereas ONOO� can induce extensive

DNA base oxidation and deamination, leading to mu-

tations [46]. Thus, agents that are able to protect against

ONOO� dependent damage may be therapeutically use-ful. Indeed, substantial attention has been directed to-

wards evaluation of natural products and dietary

constituents as antioxidant agents [21–24,26,27,29]. Al-

though the antioxidant properties ofAllium sp. have been

investigated, these efforts have largely been confined to

the flavonoid, S-Alk(en)yl-LL-cysteine sulphoxides and

thiosulphinate constituents (reviewed in [21,22]).

3-MP was identified only in onions in 1999 [30,31]and the potential antioxidant properties have yet to be

characterised. Scavenging of ONOO� by antioxidants

based on assays involving tyrosine nitration, inactiva-

tion of a1-antiproteinase [16], and inhibition of cellular

toxicity is suggested as a useful research tool providing

additional valuable information on the antioxidant

profile of the biomolecules. In each assay examined, 3-

MP significantly inhibited ONOO�-mediated toxicity atlow concentrations relative to ONOO�. Indeed, 3-MP

was significantly more effective on a molar basis than

GSH at inhibiting ONOO�-dependent tyrosine nitration

(Fig. 2) and inactivation of a1-AP (Fig. 1A). Interest-

ingly, unlike other thiol compounds we previously tested

in these experimental systems such as cysteine, penicil-

lamine, and captropil [41,42], 3-MP did not generate

secondary species capable of aggravating a1-AP inacti-vation on reaction with ONOO� (Fig. 3A). 3-MP was

also effective in cell culture experiments and greater in-

hibition was observed than with GSH. Significant inhi-

bition of ONOO�-dependent loss of cell viability was

observed at <30lM concentrations relative to high

concentrations of ONOO� (500 lM). Furthermore, in-

tracellular ROS generation and tyrosine nitration was

effectively inhibited by 3-MP and less so by GSH pre-sumably, due to the higher lipophilicity of 3-MP thus

allowing more extensive intracellular distribution.

However the extent to which 3-MP penetrates the cell

membrane and the extent to which intracellular metab-

olites contribute to 3-MPs antioxidant activity are cur-

rently being evaluated by our laboratory.

These data suggest that 3-MP has the potential to act

as an inhibitor of ONOO�-mediated processes in vivoand the antioxidant action of this novel constituent of

onions deserves further study.

Acknowledgments

We are grateful to the National Medical Research Council of

Singapore (NMRC/0474/2000 and NMRC/0481/2000) and the

National University of Singapore Academic Research Fund (R18300

0053214) for their generous research support.

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