Extended hypoxia-mediated H2 S production provides for long-term oxygen sensing.

AIM: Numerous studies have shown that H2 S serves as an acute oxygen sensor in a variety of cells. We hypothesize that H2 S also serves in extended oxygen sensing. METHODS: Here, we compare the effects of extended exposure (24-48 hours) to varying O2 tensions on H2 S and polysulphide metabolism in human embryonic kidney (HEK 293), human adenocarcinomic alveolar basal epithelial (A549), human colon cancer (HTC116), bovine pulmonary artery smooth muscle, human umbilical-derived mesenchymal stromal (stem) cells and porcine tracheal epithelium (PTE) using sulphur-specific fluorophores and fluorometry or confocal microscopy. RESULTS: All cells continuously produced H2 S in 21% O2 and H2 S production was increased at lower O2 tensions. Decreasing O2 from 21% to 10%, 5% and 1% O2 progressively increased H2 S production in HEK293 cells and this was partially inhibited by a combination of inhibitors of H2 S biosynthesis, aminooxyacetate, propargyl glycine and compound 3. Mitochondria appeared to be the source of much of this increase in HEK 293 cells. H2 S production in all other cells and PTE increased when O2 was lowered from 21% to 5% except for HTC116 cells where 1% O2 was necessary to increase H2 S, presumably reflecting the hypoxic environment in vivo. Polysulphides (H2 Sn , where n = 2-7), the key signalling metabolite of H2 S also appeared to increase in many cells although this was often masked by high endogenous polysulphide concentrations. CONCLUSION: These results show that cellular H2 S is increased during extended hypoxia and they suggest this is a continuously active O2 -sensing mechanism in a variety of cells.

Hydrogen sulfide induces heme oxygenase-1 in human kidney cells.

Hydrogen sulfide (H2S) has been shown to have a potential protective role in a number of disease states including diabetes and various kidney disorders; however, the mechanisms involved are still unclear. The aim of this study was to investigate if H2S effects the expression of the antioxidant enzyme heme oxygenase-1 (HO-1) in human kidney cells. Human mesangial cells and human podocytes were cultured at normal physiological glucose concentration (5.5 mM) and then treated with different H2S donors for a 24-h period. Protein was then extracted from the cells, and the expression levels of HO-1 determined by Western blotting. There was a significant increase in HO-1 expression after treatment with the H2S donors in both mesangial and podocyte cells. These results suggest that H2S has a role in the regulation of HO-1 expression, and the ability to upregulate this antioxidant enzyme maybe a potential mechanism by which H2S exerts its protective effects.

Lymphocytes from rheumatoid arthritis patients have elevated levels of intracellular peroxiredoxin 2, and a greater frequency of cells with exofacial peroxiredoxin 2, compared with healthy human lymphocytes.

Peroxiredoxin 2 has immune regulatory functions, but its expression in human peripheral blood lymphocytes and levels in extracellular fluid in healthy subjects and rheumatoid arthritis patients are poorly described. In the present study, the median intracellular peroxiredoxin 2 protein content of lymphocytes from rheumatoid arthritis patients was more than two-fold higher compared with healthy subjects' lymphocytes. Intracellular peroxiredoxin 3 levels were similar in healthy and rheumatoid arthritis lymphocytes. Flow cytometry detected peroxiredoxin 2 on the surface of ca. 8% of T cells and ca. 56% of B cells (median % values) of all subjects analyzed. Exofacial thioredoxin-1 was also observed. In the total lymphocyte population from rheumatoid arthritis patients, few cells (median, 6%) displayed surface peroxiredoxin 2. In contrast, a significantly increased proportion of interleukin-17(+ve) lymphocytes were exofacially peroxiredoxin 2(+ve) (median, 39%). Prdx2 was also detected in human extracellular fluids. We suggest that crucial inflammatory cell subsets, i.e. interleukin-17(+ve) T cells, exhibit increased exofacial redox-regulating enzymes and that peroxiredoxin 2 may be involved in the persistence of pro-inflammatory cells in chronic inflammation.

Imaging the uptake of gold nanoshells in live cells using plasmon resonance enhanced four wave mixing microscopy.

Gold nanoshells (GNS) are novel metal nanoparticles exhibiting attractive optical properties which make them highly suitable for biophotonics applications. We present a novel investigation using plasmon-enhanced four wave mixing microscopy combined with coherent anti-Stokes Raman scattering (CARS) microscopy to visualize the distribution of 75 nm radius GNS within live cells. During a laser tolerance study we found that cells containing nanoshells could be exposed to < 2.5 mJ each with no photo-thermally induced necrosis detected, while cell death was linearly proportional to the power over this threshold. The majority of the GNS signal detected was from plasmon-enhanced four wave mixing (FWM) that we detected in the epi-direction with the incident lasers tuned to the silent region of the Raman spectrum. The cellular GNS distribution was visualized by combining the epi-detected signal with forwards-detected CARS at the CH2 resonance. The applicability of this technique to real-world nanoparticle dosing problems was demonstrated in a study of the effect of H2S on nanoshell uptake using two donor molecules, NaHS and GYY4137. As GYY4137 concentration was increased from 10 µM to 1 mM, the nanoshell pixel percentage as a function of cell volume (PPCV) increased from 2.15% to 3.77%. As NaHS concentration was increased over the same range, the nanoshell PPCV decreased from 12.67% to 11.47%. The most important factor affecting uptake in this study was found to be the rate of H2S release, with rapid-release from NaHS resulting in significantly greater uptake.

Hydrogen sulfide effects on stomatal apertures.

Hydrogen sulfide (H(2)S) has recently been reported to be a signaling molecule in plants. It has been well established that is has such roles in animals and it has been suggested that it is included into the group of gasotransmitters. We have recently shown that hydrogen sulfide causes stomatal opening in the model plant Arabidopsis thaliana. H(2)S can be supplied to the plant tissues from donors such as sodium hydrosulfide (NaSH) or more recently from slow release H(2)S donor molecules such as GYY4137. Both give similar effects, that is, they cause stomatal opening. Furthermore both H(2)S donors reduced the accumulation of nitric oxide (NO) induced by abscisic acid (ABA) treatment of leaf tissues. Here similar work has been repeated in a crop plant, Capsium anuum, and similar data has been obtained, suggesting that such effects of hydrogen sulfide on plants is not confined to model species.

Hydrogen sulfide and its possible roles in myocardial ischemia in experimental rats.

The role of hydrogen sulfide (H(2)S) in myocardial infarction (MI) has not been previously studied. We therefore investigated the effect of H(2)S in a rat model of MI in vivo. Animals were randomly divided into three groups (n = 80) and received either vehicle, 14 micromol/kg of sodium hydrosulfide (NaHS), or 50 mg/kg propargylglycine (PAG) everyday for 1 wk before surgery, and the treatment was continued for a further 2 days after MI when the animals were killed. The mortality was 35% in vehicle-treated, 40% in PAG-treated, and 27.5% in NaHS-treated (P < 0.05 vs. vehicle) groups. Infarct size was 52.9 +/- 3.5% in vehicle-treated, 62.9 +/- 7.6% in PAG-treated, and 43.4 +/- 2.8% in NaHS-treated (P < 0.05 vs. vehicle) groups. Plasma H(2)S concentration was significantly increased after MI (59.2 +/- 7.16 microM) compared with the baseline concentration (i.e., 38.2 +/- 2.07 microM before MI; P < 0.05). Elevated plasma H(2)S after MI was abolished by treatment of animals with PAG (39.2 +/- 5.02 microM). We further showed for the first time cystathionine-gamma-lyase protein localization in the myocardium of the infarct area by using immunohistochemical staining. In the hypoxic vascular smooth muscle cells, we found that cell death was increased under the stimuli of hypoxia but that the increased cell death was attenuated by the pretreatment of NaHS (71 +/- 1.2% cell viability in hypoxic vehicle vs. 95 +/- 2.3% in nonhypoxic control; P < 0.05). In conclusion, endogenous H(2)S was cardioprotective in the rat model of MI. PAG reduced endogenous H(2)S production after MI by inhibiting cystathionine-gamma-lyase. The results suggest that H(2)S might provide a novel approach to the treatment of MI.

IKKbeta programs to turn on the GADD45alpha-MKK4-JNK apoptotic cascade specifically via p50 NF-kappaB in arsenite response.

Cross talk between NF-kappaB and c-Jun N-terminal kinases (JNKs) has been implicated in the cell life and death decision under various stresses. Functional suppression of JNK activation by NF-kappaB has recently been proposed as a key cellular survival mechanism and contributes to cancer cells escaping from apoptosis. We provide a novel scenario of the proapoptotic role of IkappaB kinase beta (IKKbeta)-NF-kappaB, which can act as the activator of the JNK pathway through the induction of GADD45alpha for triggering MKK4/JNK activation, in response to the stimulation of arsenite, a cancer therapeutic reagent. This effect of IKKbeta-NF-kappaB is dependent on p50 but not the p65/relA NF-kappaB subunit, which can increase the stability of GADD45alpha protein through suppressing its ubiquitination and proteasome-dependent degradation. IKKbeta-NF-kappaB can therefore either activate or suppress the JNK cascade and consequently mediate pro- or antiapoptotic effects, depending on the manner of its induction. Furthermore, the NF-kappaB p50 subunit can exert a novel regulatory function on protein modification independent of the classical NF-kappaB transcriptional activity.

Cardioprotective effects of nitroparacetamol and paracetamol in acute phase of myocardial infarction in experimental rats.

We aimed to determine whether nitroparacetamol (NO-paracetamol) and paracetamol exhibit cardioprotective effects. Myocardial infarction (MI) was induced in rats, and drug treatment was started 1 wk before surgery. Mortality rate and infarct size at 2 days after MI were compared. Treatment groups included vehicle (saline), paracetamol (5 mg x kg(-1) x day(-1)) and NO-paracetamol (15 mg x kg(-1) x day(-1)). Mortality rates for vehicle (n = 80), paracetamol (n = 79), and NO-paracetamol (n = 76) groups were 37.5%, 21.5%, and 26.3%, respectively. Infarct size for the vehicle group was 44.8% (+/-6.1%) of the left ventricle (LV). For the paracetamol and NO-paracetamol groups, infarct size was 31.3% (+/-5.6%) and 30.7% (+/-8.1%) of the LV, respectively. Both paracetamol- and NO-paracetamol-treated groups showed increased activities of catalase and SOD compared with the vehicle group. They could attenuate endothelial, inducible, and neuronal nitric oxide synthase and cyclooxygenase-1 and -2 gene expression after MI. The observation indicates the potential clinical significance of the cardioprotective effects of these drugs.

Bioactive S-alk(en)yl cysteine sulfoxide metabolites in the genus Allium: the chemistry of potential therapeutic agents.

S-Alk(en)yl cysteine sulfoxides are odourless, non-protein sulfur amino acids typically found in members of the family Alliaceae and are the precursors to the lachrymatory and flavour compounds found in the agronomically important genus Allium. Traditionally, Allium species, particularly the onion (Allium cepa) and garlic (A. sativum), have been used for centuries in European, Asian and American folk medicines for the treatment of numerous human pathologies, however it is only recently that any significant progress has been made in determining their mechanisms of action. Indeed, our understanding of the role of Allium species in human health undoubtedly comes from the combination of several academic disciplines including botany, biochemistry and nutrition. During tissue damage, S-alk(en)yl cysteine sulfoxides are converted to their respective thiosulfinates or propanethial-S-oxide by the action of the enzyme alliinase (EC 4.4.1.4). Depending on the Allium species, and under differing conditions, thiosulfinates can decompose to form additional sulfur constituents including diallyl, methyl allyl, and diethyl mono-, di-, tri-, tetra-, penta-, and hexasulfides, the vinyldithiins and (E)- and (Z)-ajoene. Recent reports have shown onion and garlic extracts, along with several principal sulfur constituents, can induce phase II detoxification enzymes like glutathione-S-transferases (EC 2.5.1.18) and quinone reductase (QR) NAD(P)H: (quinine acceptor) oxidoreductase (EC 1.6.99.2) in mammalian tissues, as well as also influencing cell cycle arrest and apoptosis in numerous in vitro cancer cell models. Moreover, studies are also beginning to highlight a role of Allium-derived sulfur compounds in cardiovascular protection. In this review, we discuss the chemical diversity of S-alk(en)yl cysteine sulfoxide metabolites in the context of their biochemical and pharmacological mechanisms.

Hydrogen sulfide protects colon cancer cells from chemopreventative agent beta-phenylethyl isothiocyanate induced apoptosis.

AIM: Hydrogen sulfide (H(2)S) is a prominent gaseous constituent of the gastrointestinal (GI) tract with known cytotoxic properties. Endogenous concentrations of H(2)S are reported to range between 0.2-3.4 mmol/L in the GI tract of mice and humans. Considering such high levels we speculate that, at non-toxic concentrations, H(2)S may interact with chemical agents and alter the response of colonic epithelium cells to such compounds. The GI tract is a major site for the absorption of phytochemical constituents such as isothiocyanates, flavonoids, and carotenoids, with each group having a role in the prevention of human diseases such as colon cancer. The chemopreventative properties of the phytochemical agent beta-phenyethyl isothiocyanate (PEITC) are well recognized. However, little is currently known about the physiological or biochemical factors present in the GI tract that may influence the biological properties of ITCs. The current study was undertaken to determine the effects of H(2)S on PEITC mediated apoptosis in colon cancer cells. METHODS: Induction of apoptosis by PEITC in human colon cancer HCT116 cells was assessed using classic apoptotic markers namely SubG1 population analysis, caspase-3 like activity and nuclear fragmentation and condensation coupled with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide) viability assay and LDH leakage. RESULTS: PEITC significantly induced apoptosis in HCT116 cells as assessed by SubG1 population formation, nuclear condensation, LDH leakage and caspase-3 activity after 24 h, these data being significant from control groups (P<0.01). In contrast, co-treatment of cells with physiological concentrations of H2S (0.1-1 mmol/L) prevented PEITC mediated apoptosis as assessed using the parameters described. CONCLUSION: PEITC effectively induced cell death in the human adenocarcinoma cell line HCT116 in vitro through classic apoptotic mechanisms. However, in the presence of H(2)S, apoptosis was abolished. These data suggest that H(2)S may play a significant role in the response of colonic epithelial cells to beneficial as well as toxic agents present within the GI tract.

Broccoli and watercress suppress matrix metalloproteinase-9 activity and invasiveness of human MDA-MB-231 breast cancer cells.

A high dietary intake of cruciferous vegetables has been associated with a reduction in numerous human pathologies particularly cancer. In the current study, we examined the inhibitory effects of broccoli (Brassica oleracea var. italica) and watercress (Rorripa nasturtium aquaticum) extracts on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cancer cell invasion and matrix metalloproteinase-9 activity using human MDA-MB-231 breast cancer cells. Aberrant overexpression of matrix metalloproteinases, including metalloproteinase-9, is associated with increased invasive potential in cancer cell lines. Our results demonstrate that extracts of broccoli and Rorripa suppressed TPA-induced MMP-9 activity and invasiveness in a concentration dependent manner as determined by zymographic analysis. Furthermore, fractionation of individual extracts followed by liquid chromatography mass spectroscopy analysis (LC-MS) revealed that the inhibitory effects of each vegetable were associated with the presence of 4-methysulfinylbutyl (sulforaphane) and 7-methylsulphinylheptyl isothiocyanates. Taken together, our data indicate that isothiocyanates derived form broccoli and Rorripa inhibit metalloproteinase 9 activities and also suppress the invasive potential of human MDA-MB-231 breast cancer cells in vitro. The inhibitory effects observed in the current study may contribute to the suppression of carcinogenesis by diets high in cruciferous vegetables.

Beta-phenylethyl and 8-methylsulphinyloctyl isothiocyanates, constituents of watercress, suppress LPS induced production of nitric oxide and prostaglandin E2 in RAW 264.7 macrophages.

Beta-phenylethyl (PEITC) and 8-methylsulphinyloctyl isothiocyanates (MSO) represent two phytochemical constituents present in watercress Rorripa nasturtium aquaticum, with known chemopreventative properties. In the present investigation, we examined whether PEITC and MSO could modulate the inflammatory response of Raw 264.7 macrophages to bacterial lipopolysaccharide (LPS) by assessment of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Overproduction of both nitric oxide (NO) and prostaglandins (PGE) has been associated with numerous pathological conditions including chronic inflammation and cancer. Our results demonstrate that LPS (1 microg/ml approximately 24 h) induced nitrite and prostaglandin E2 (PGE-2) synthesis in Raw 264.7 cells was attenuated by both isothiocyanates (ITCs) in a concentration-dependent manner. Both PEITC and MSO decreased (iNOS) and (COX-2) protein expression levels leading to reduced secretion of both pro-inflammatory mediators. Interestingly, the reduction in both iNOS and COX-2 expression were associated with the inactivation of nuclear factor-kappaB and stabilization of IkappaBalpha. Taken together our data gives further insight into the possible chemopreventative properties of two dietary derived isothiocyanates from watercress.

Effects of purified herbal extract of Salvia miltiorrhiza on ischemic rat myocardium after acute myocardial infarction.

In the current study, we compared purified Salvia miltiorrhiza extract (PSME) with Angiotensin-converting enzyme inhibitor, Ramipril, in in vitro experiments and also in vivo using animal model of myocardial infarction. PSME was found to have a significantly higher trolox equivalent antioxidant capacity which indicated a great capacity for scavenging free radicals. PSME could also prevent pyrogallo red bleaching and DNA damage. After 2 weeks treatment with PSME or Ramipril, survival rates of rats with experimental myocardial infarction were marginally increased (68.2% and 71.4%) compared with saline (61.5%). The ratios of infarct size to left ventricular size in both PSME-and Ramipril-treated rats were significantly less than that in the saline-treated group. Activity of cardiac antioxidant enzyme superoxide dismutase (SOD) was significant higher while level of Thiobarbituric acid-reactive substances (TBARs) was lower in the PSME treated group. Purified and standardized Chinese herb could provide an alternative regimen for the prevention of ischemic heart disease.

Protective effects of Asian green vegetables against oxidant induced cytotoxicity.

AIM: To evaluate the antioxidant and phase II detoxification enzyme inducing ability of green leaf vegetables consumed in Asia. METHODS: The antioxidant properties of six commonly consumed Asian vegetables were determined using the ABTS, DPPH, deoxyribose, PR bleaching and iron- ascorbate induced lipid peroxidation assay. Induce of phase II detoxification enzymes was also determined for each respective vegetable extract. Protection against authentic ONOO- and HOCl mediated cytotoxicity in human colon HCT116 cells was determined using the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide) viability assay. RESULTS: All of the extracts derived from green leaf vegetables exhibited antioxidant properties, while also having cytoprotective effects against ONOO- and HOCl mediated cytotoxicity. In addition, evaluation of the phase II enzyme inducing ability of each extract, as assessed by quinone reductase and glutathione-S-transferase activities, showed significant variation between the vegetables analyzed. CONCLUSION: Green leaf vegetables are potential sources of antioxidants and phase II detoxification enzyme inducers in the Asian diet. It is likely that consumption of such vegetables is a major source of beneficial phytochemical constituents that may protect against colonic damage.