Effect of naturally occurring organosulfur compounds on nitric oxide production in lipopolysaccharide-activated macrophages

doi:10.1016/S0024-3205(02)01685-5

Life Sciences

Volume 71, Issue 4, 14 June 2002, Pages 411-419

https://doi.org/10.1016/S0024-3205(02)01685-5 Get rights and content

Abstract

Excessive nitric oxide (NO) production is involved in cellular injury and possibly in the multistage process of carcinogenesis. In this study, we investigated the effect of organosulfur compounds (S-allyl cysteine, allyl sulfide, diallyl disulfide, allyl isothiocyanate, phenyl isothiocyanate, and benzyl isothiocyanate) that are found in allium or cruciferous vegetables on NO production in J774.1 macrophages activated with lipopolysaccharide (LPS). Diallyl disulfide, allyl, phenyl, and benzyl isothiocyanates inhibited NO production, as evaluated by nitrite formation at 25 μM. Allyl and benzyl isothiocyanates, the most active of the six organosulfur compounds, exhibited dose-dependent inhibition and had IC₅₀ values of 1.6 and 2.7 μM, respectively. Western blot analysis suggested that suppression of the induction of inducible NO synthase (iNOS) expression is responsible for the inhibition of NO production by allyl and benzyl isothiocyanates. In contrast, these isothiocyanates increased LPS-stimulated tumor necrosis factor alpha (TNF-α) release, suggesting their selective action on genes activated by LPS. Our results demonstrate that certain organosulfur compounds inhibit NO synthesis in LPS–activated macrophages, and the inhibitory effect may be a significant component of their anticarcinogenic activity.

Introduction

Nitric oxide (NO), a reactive free radical gas, is synthesized in vivo by a family of constitutively expressed and inducible nitric oxide synthases (NOS) [1], [2], [3]. As a signal messenger, NO produced by the NOS isoform present in endothelial cells (eNOS) is involved in blood pressure control through regulation of smooth muscle cell relaxation and vasodilation. The inducible NOS isoform present in macrophages (iNOS) activated with some cytokines or lipopolysaccharide (LPS) generates a large quantity of NO, which is partially responsible for the tumoricidal, bactericidal, and possibly immune regulatory activities. Excessive NO production by up-regulation of iNOS during chronic infection or inflammation has been implicated in cancer development. It has been proposed that reactive nitrogen species (RNS), such as NO and its derivatives, e.g. peroxynitrite (ONOO⁻) and nitrogen dioxide (NO₂), cause DNA or tissue damage, contributing to the multistage carcinogenesis process [4].

Epidemiological studies suggest that frequent consumption of allium and cruciferous vegetables may be associated with a lower incidence of cancers [5], [6]. Organosulfur compounds derived in these vegetables, such as sulfides and isothiocyanates, have been reported to possess anticarcinogenic properties [7], [8]. Wargovich et al. [5] found that administering allyl sulfide present in garlic to rats during their exposure to the colon carcinogen, dimethylhydrazine, or the esophageal carcinogen, N-nitrosomethylbenzylamine, reproducibly inhibited neoplasia to a significant degree. Wattenberg described that various isothiocyanates prevent 7,12-dimethylbenz[a]anthracene (DMBA)-induced mouse mammary tumorigenesis [9]. These organosulfur compounds are able to influence phase 1 and phase 2 biotransformation enzyme activities, thereby possibly influencing several processes related to chemical carcinogenesis, e.g. the metabolism, DNA-binding, and mutagenic activity of promutagens. The anticarcinogenic activity of organosulfur compounds is considered to be due to their modulatory action on these detoxification enzymes [5], [8], [10].

The facts mentioned above focused our interest on the question of whether these anticarcinogenic organosulfur compounds have an effect on inducible nitric oxide synthase (iNOS), which is thought to be related to the carcinogenesis. In the present study, the effect of these organosulfur compounds on NO synthesis in LPS-activated macrophages was investigated.

Section snippets

Reagents

S-allyl cysteine, allyl sulfide, and diallyl disulfide were from Tokyo Chemical Industry (Tokyo, Japan). Allyl isothiocyanate, sulfanilamide, and N-1-naphthylethylenediamine dihydrochloride were from Wako Pure Chemical Industries (Osaka, Japan). Phenyl isothiocyanate, benzyl isothiocyanate, LPS (Escherichia coli, O111:B4), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and anti-α-tubulin mouse antibody were from Sigma-Aldrich (MO, USA). RPMI1640 medium was from Nissui

Nitrite assay

Nitrite (a breakdown product of NO) in culture supernatants was measured by adding 100 μl of Griess reagent (1% sulfanilamide and 0.1% N-1-naphthylethylenediamine dihydrochloride in 2.5% H₃PO₄) to 100 μL samples of medium [11]. The optical density at 550 nm (OD₅₅₀, reference wavelength 655 nm) was measured by using a microplate reader (Bio-Rad Laboratories, CA, USA). Nitrite concentrations were calculated by comparison with OD₅₅₀ of standard solutions of sodium nitrite.

MTT assay

Mitochondrial respiration, an indicator of cell viability, was assessed by the mitochondria-dependent reduction of MTT to formazan [12]. Cells were incubated with MTT (0.2 mg/ml) for 1 h at 37 °C. Cell medium was removed, and cells were solubilized in dimethyl sulfoxide. The extent of reduction of MTT to formazan within cells was quantified by measurement of OD₅₅₀ using a microplate reader.

Western blotting

J774.1 cells were scraped off the culture dishes and washed with cold PBS. The cells were pelleted by centrifugation at 4 °C and homogenized in extraction buffer (25 mM NaH₂PO₄, 75 mM NaCl, 5 mM EDTA, 1% Triton X-100, 100 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 20 μg/ml aprotinin, pH 7.4). The lysate was centrifuged at 15,000 g for 30 min at 4 °C. Protein concentration of the supernatant was determined by the bicinchoninic acid protein assay (Pierce, IL,

TNF-α

Tumor necrosis factor alpha (TNF-α) in culture supernatant was measured using a quantitative sandwich enzyme immunoassay (R&D Systems, MN, USA). The assay was performed according to the manufacture's instruction.

Statistical analysis

Each experiment (Table 1, Table 2 and Fig. 2) was performed in triplicate, and the results are expressed as the arithmetic mean ± standard deviation. Statistical analysis of the data was performed by student's t-test.

Effect of organosulfur compounds on NO production

The effect of organosulfur compounds (25 μM, Fig. 1) that are found in allium or cruciferous vegetables on NO production, measured as nitrite by the Griess reaction, in J774.1 macrophages activated with LPS (10 μg/ml) for 24 h is shown in Table 1.

In sulfides, only diallyl disulfide reduced NO production; whereas all isothiocyanates tested in this assay exhibited the inhibitory effect on LPS-induced NO production. Of the six organosulfur compounds, allyl and benzyl isothiocyanates significantly

Discussion

Organosulfur compounds derived from allium or cruciferous vegetables possess anticarcinogenic activity. The modulatory action of these organosulfur compounds on phase 1 and phase 2 detoxification enzyme activities has been reported to be related to their mechanism for anticarcinogenic activity [5], [8], [10].

In this present study, we clearly showed that certain organosulfur compounds suppress NO production in LPS-activated J774.1 macrophages. Of the compounds tested, allyl and benzyl

Acknowledgments

This study was supported by grant-in-aid from the Ministry of Agriculture, Forestry and Fisheries of Japan.

References (21)

D.T. Verhoeven et al.
A review of mechanisms underlying anticarcinogenicity by brassica vegetables
Chem. Biol. Interact.
(1997)
G.S. Stoewsand
Bioactive organosulfur phytochemicals in Brassica oleracea vegetables—a review
Food Chem. Toxicol.
(1995)
L.C. Green et al.
Analysis of nitrate, nitrite, and [¹⁵N]nitrate in biological fluids
Anal. Biochem.
(1982)
C. Szabó et al.
Dihydropyridine antagonists and agonists of calcium channels inhibit the induction of nitric oxide synthase by endotoxin in cultured macrophages
Biochem. Biophys. Res. Commun.
(1993)
Z.H. Németh et al.
Effect of the phosphodiesterase III inhibitor amrinone on cytokine and nitric oxide production in immunostimulated J774.1 macrophages
Eur. J. Pharmacol.
(1997)
V.M. Dirsch et al.
Effect of allicin and ajoene, two compounds of garlic, on inducible nitric oxide synthase
Atherosclerosis
(1998)
M.M. Borges et al.
Prostaglandin and nitric oxide regulate TNF-α production during Trypanosoma cruzi infection
Immunol. Lett.
(1998)
T.L. Wadsworth et al.
Effects of the wine polyphenolics quercetin and resveratrol on pro-inflammatory cytokine expression in RAW 264.7 macrophages
Biochem. Pharmacol.
(1999)
J.H. Steer et al.
Glucocorticoids suppress tumor necrosis factor-α expression by human monocytic THP-1 cells by suppressing transactivation through adjacent NF-κB and c-Jun-activating transcription factor-2 binding sites in the promoter
J. Biol. Chem.
(2000)
S. Moncada et al.
Nitric oxide: physiology, pathophysiology, and pharmacology
Pharmacol. Rev.
(1991)

There are more references available in the full text version of this article.

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Effect of naturally occurring organosulfur compounds on nitric oxide production in lipopolysaccharide-activated macrophages

Abstract

Introduction

Section snippets

Reagents

Nitrite assay

MTT assay

Western blotting

TNF-α

Statistical analysis

Effect of organosulfur compounds on NO production

Discussion

Acknowledgments

Chem. Biol. Interact.

Food Chem. Toxicol.

Anal. Biochem.

Biochem. Biophys. Res. Commun.

Eur. J. Pharmacol.

Atherosclerosis

Immunol. Lett.

Biochem. Pharmacol.

J. Biol. Chem.

Nitric oxide: physiology, pathophysiology, and pharmacology

Pharmacol. Rev.