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