4(α-L-RHAMNOSYLOXY)-BENZYL ISOTHIOCYANATE, A BIOACTIVE PHYTOCHEMICAL THAT DEFENDS CEREBRAL TISSUE AND PREVENTS SEVERE DAMAGE INDUCED BY FOCAL ISCHEMIA/REPERFUSION.

Natural compounds are a promising source to treat several pathologies. The present study shows the in vivo pharmacological beneficial effect of 4(α-L-rhamnosyloxy)-benzyl isothiocyanate (glucomoringin isothiocyanate; GMG-ITC) obtained from glucomoringin (GMG; 4(α;-L-rhamnosyloxy)- benzyl glucosinolate), purified from Moringa oleifera seeds and hydrolyzed by myrosinase enzyme (β-thioglucoside glucohydrolase; E.C. 3.2.1.147). Cerebral ischemia/reperfusion (CIR) was induced in rats according to a classic model of carotid artery occlusion for a time period of 1 h and the reperfusion time was prolonged for seven days. GMG-ITC (3.5 mg GMG/ml plus 30 µl enzyme/rat; one ml i.p./rat) was administered 15 min after the beginning of ischemia and daily. The results clearly show that GMG-ITC possesses the capability to counteract the CIR-induced damage reducing TNF-alpha release, IκB-alpha cytosolic degradation/NFκBp65 nuclear translocation, as well as several other direct or indirect markers of inflammation (phospho-ERK p42/44, p-selectin) and oxidative stress (inducible Nitric Oxide Synthase (iNOS), MMP-9). GMG-ITC was shown to exert neuroprotective properties in preventing CIR-induced damage and the related cascade of inflammatory and oxidative mediators that exacerbate the progression of this disease in an experimental rat model. Our results clearly show that the tested phytochemical GMG-ITC possesses the capability to counteract CIR-induced damage.

The protective effects of bioactive (RS)-glucoraphanin on the permeability of the mice blood-brain barrier following experimental autoimmune encephalomyelitis.

OBJECTIVES: Alterations in blood-brain barrier (BBB) permeability are due to the disruption of the Tight Junctions (TJs), large multiprotein complexes important for the maintenance of structural integrity and for permeability of the barrier. In this experimental study we evaluated the neuroprotective role of (RS)-glucoraphanin, a glucosinolate present in Brassicaceae, notably in Tuscan black kale, and bioactivated with myrosinase enzyme (bioactive RS-GRA) (10 mg/kg/d intraperitoneally), to prevent the dysfunction of BBB, in an experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). MATERIALS AND METHODS: EAE was induced by immunization with myelin oligodendroglial glycoprotein peptide (MOG)35-55 in mice. By western blot analysis of brain tissues, we evaluated expression and distribution of the TJ-associated proteins, claudin-1, -3, -5 and ZO-1. Additionally, in order to gain a better insight into the mechanisms of action of bioactive RS-GRA, we investigated Foxp3, ERK1/2 and caspase 3 expression associated both to inflammatory response as well as to apoptotic pathway. RESULTS: Our results demonstrated that treatment with bioactive RS-GRA counteracts the alteration of all these parameters and preserves TJ integrity through an antinflammatory and antiapoptotic activity during MS. CONCLUSIONS: Bioactive RS-GRA, could be a therapeutic perspective helpful in preventing dysfunction of the BBB.

Structural influence of isothiocyanates on expression of cytochrome P450, phase II enzymes, and activation of Nrf2 in primary rat hepatocytes.

Primary cultures of rat hepatocytes were used to investigate whether and how eight isothiocynates (ITCs) with different chemical structures (the aromatic benzyl, 4-hydroxybenzyl, phenethyl isothiocyanates and the aliphatic allyl, napin, iberin, raphasatin isothiocyanates and sulforaphane) derived from hydrolyzed glucosinolates, were able to modulate cytochrome P450 (CYP) and antioxidant/detoxifying enzymes and to activate the Nrf2 transcription factor. The aromatic ITCs at 40 µM markedly increased the transcription of CYP1A1 and 1A2 mRNA and increased the associated ethoxyresorufin O-deethylase (EROD) activity after 24 h of treatment. By contrast, the aliphatic ITCs (40 µM) decreased CYP1A1 and 1A2 transcription, together with the corresponding EROD activity. The same treatment also caused a striking and similar transcriptional repression of CYP3A2, and the corresponding benzyloxyquinoline debenzylase activity in response to all the ITCs tested. In the same culture conditions, most of the antioxidant/detoxifying enzymes were significantly up-regulated by 40µM ITCs. In particular, NAD(P)H:quinone oxidoreductase and heme oxygenase-1 were induced, although to different levels, at transcriptional, protein and/or activity levels by all the ITCs. However, glutathione S-transferase activity was not induced by the allyl, benzyl, and 4-hydroxybenzyl ITCs, glutathione reductase activity was not induced by benzyl, and 4-hydroxybenzyl ITCs and catalase activity was not induced by allyl ITC. As for the Nrf2 transcription factor, a partial translocation of its protein from the cytosol to the nucleus was revealed by immunoblotting after 1h of treatment for all the ITCs tested. The ability of ITCs to induce the antioxidant and phase II enzymes did not appear to be affected by their hydrophilicity or other structural factors. Taken together, these results show that these ITCs are effective inducers of ARE/Nrf2-regulated antioxidant/detoxifying genes and have the potential to inhibit, at least in rat liver, the bioactivation of carcinogens dependent on CYP3A2 catalysis.

Is cholangiocarcinoma another complication of insulin resistance: a report of three cases.

BACKGROUND: Cholangiocarcinoma is the second most common primary liver cancer, and the number of cases of intrahepatic cholangiocarcinoma (ICC) have been steadily increasing worldwide. Although the reasons for this surge are unknown, insulin resistance (IR) could be a risk factor, similar to what has been reported for other cancers. CASE REPORT: We report on 3 cases of ICC arising in subjects sharing IR as an underlying risk factor. Case 1 was an obese and dyslipidemic patient with NAFLD. The second and the third patients were affected by type 2 diabetes. CONCLUSIONS: Evidence for a link between IR and onset of cholangiocarcinoma in our patients rests on three lines of evidence: epidemiological, biological, and exclusion of others risk factors. Studies are needed to confirm our hypothesis that IR is a risk factor for the development of ICC.

A mixture of isothiocyanates induces cyclin B1- and p53-mediated cell-cycle arrest and apoptosis of human T lymphoblastoid cells.

As with other candidate chemopreventive agents, most of our knowledge on the biological effects of isothiocyanates (the many sulfur-containing metabolites found in cruciferous vegetables) comes from studies of single natural or synthetic compounds. To investigate whether the biological/chemopreventive effects of administration of single isothiocyanates can differ from those of a mixture of isothiocyanates, we tested the effects of a mixture of four different isothiocyanates on cell-cycle progression and apoptosis in human T leukemia Jurkat cells, and identified some of the molecular pathways triggered by the mixture. The mixture affected critical points of the cell cycle via modulation of the expression of cyclin B1. Moreover, it induced apoptosis, mediated by an increase in p53 and bax (expression of bcl-2 was unaffected). Comparison of the data with those previously obtained with the single isothiocyanates under identical experimental conditions provides evidence that the quantitative effects of a single, specific isothiocyanate can be significantly different from those of an isothiocyanate mixture at realistic doses.

Genetic and metabolic effects of gluconasturtiin, a glucosinolate derived from cruciferae.

It is thought that induction of detoxifying phase-II drug metabolizing enzymes or inhibition of bioactivating phase-I by phytoalexins could protect against mutagens and neoplasia. In the search for potential naturally occurring molecular chemoprevention agents, particular attention has been devoted to isothiocyanates, which are breakdown products-via myrosinase-of glucosinolates such as gluconasturtiin (GNST), a natural constituent of cruciferae. Here, we first investigated the ability of GNST to modulate metabolizing enzymes in male Swiss Albino CD1 mice injected by gavage (24 mg/kg or 48 mg/kg b.w.) with GNST either in single or repeated (daily for four consecutive days) dose. Using selected probes to various cytochrome P450 (CYP) isoforms, a marked and generalized decrease of CYP content, NADPH-(CYP)-c-reductase and various CYP-linked monooxygenases (measuring CYP1A1, CYP2B1/2, CYP3A1/2, CYP1A2 and CYP2E1), was observed in hepatic, renal and pulmonary subcellular preparations (up to approximately 66% loss, liver). Similar behavior was recorded using the regio- and stereo-selective hydroxylation of testosterone as multibiomarker (CYP2A1 and CYP2B9, up to approximately 96% loss), as well as with the phase-II marker glutathione S-transferase (up to approximately 50% loss, liver). We also performed genotoxicity investigations, using the diploid D7 strain of yeast Saccharomyces cerevisiae as a biological test system. GNST was able to significantly induce point reverse mutation in growing cells without myrosinase, thus suggesting either a direct GNST or a CYP-linked metabolite role in the genotoxic response. On the contrary, in suspension test, the addition of myrosinase significantly increased mitotic gene conversion, probably due to the formation of GNST-derived phenylethyl isothiocyanate (PEITC) breakdown product. Taken together, our data suggest that GNST exerts a dual effect: while strongly inhibiting the microsomal (bioactivating) metabolism, GNST also possesses genotoxic activity. This concomitant mutagenic activity underlines the necessity of overall toxicological characterization of this (or any other molecule) prior to mass chemopreventive use.

Cyclin D3 and p53 mediate sulforaphane-induced cell cycle delay and apoptosis in non-transformed human T lymphocytes.

Despite experimental evidence that sulforaphane can exert chemopreventive effects, whether these effects are specific for neoplastic cells is not known. Following our previous demonstration that sulforaphane induces cell cycle arrest and apoptosis in human T lymphoblastoid Jurkat leukemia cells and increases p53 and bax protein expression, we tested sulforaphane on non-transformed phytohemagglutinin-stimulated human lymphocytes. Here, we demonstrate that sulforaphane arrested cell cycle progression in G, phase, through a decrease in the protein expression of cyclin D3. Moreover, sulforaphane induced apoptosis (and also necrosis), mediated by an increase in the expression of p53. These findings suggest that sulforaphane is a growth modulator for T cells. Our in vitro evidence that sulforaphane is active and even cytotoxic in normal as well as transformed lymphocytes raises important questions regarding its suitability for cancer chemoprevention.

Barbarea verna as a source of 2-phenylethyl glucosinolate, precursor of cancer chemopreventive phenylethyl isothiocyanate.

The isolation of gram-amounts of 2-phenylethyl glucosinolate (gluconasturtiin, GST) from Barbarea verna seeds is reported for the first time. This vegetable source was of crucial importance to isolate GST with a high purity grade and in high yield. Indeed, B. verna seeds contain GST as the only glucosinolate, unlike other sources. The availability at low cost of GST will allow further studies to explain the claimed anticancer activity of its derived phenylethyl isothiocyanate.

Soluble and total myrosinase activity in defatted Crambe abyssinica meal.

Crambe defatted meal contains 4-6% w/w of glucosinolates, with epiprogoitrin accounting for >90% of the total. This feature limits the use of the meal as feed due to the antinutritional properties of myrosinase-glucosinolate breakdown products. In this context, myrosinase activity assumes particular importance. In this study the total and soluble myrosinase activities have been evaluated directly on defatted meals of eight Crambe abyssinica varieties. The pH-stat method, which is the most suitable for assays in heterogeneous solid-water systems, was used. The total myrosinase activity in C. abyssinica varieties, determined using epiprogoitrin as substrate, ranged from 288 to 653 units g(-1). These activity values were up to 26 times higher than those obtained using other substrates, namely, sinigrin, glucosinalbin, glucotropaeolin, progoitrin, and glucoraphenin. Crambe myrosinase is unusual in that, unlike other Brassicaceae containing a typical main glucosinolate, it does not show the same specificity toward its natural substrates.

In vitro antiproliferative activity of isothiocyanates and nitriles generated by myrosinase-mediated hydrolysis of glucosinolates from seeds of cruciferous vegetables.

A comparison of the effect of isothiocyanates and nitriles derived from some glucosinolates, namely, epi-progoitrin, sinalbin, glucotropaeolin, glucocheirolin, and glucoraphenin, on human erythroleukemic in vitro cultured cells was studied. Many studies have in fact evidenced that a consumption of vegetable containing glucosinolates could reduce the development of colorectal cancer. In the experimental conditions used, the production of isothiocyanates and nitriles from glucosinolates is almost quantitative as confirmed by HPLC or GC-MS analysis. The obtained results demonstrated that in general nitriles are considerably less potent than the corresponding isothiocyanates in inhibiting cancer cell growth. Particularly, the isothiocyanates inhibitory activity on K562 cells growth is higher in the case of products derived from epi-progoitrin, glucotropaeolin, glucoraphenin, and glucocheirolin; while for nitriles the higher activity in inhibiting K562 cells growth is showed by sinalbin-derived product. Considering the antiproliferative activity found for isothiocyanates and nitriles, further studies will be aimed to the possible application of glucosinolate-derived products as chemopreventive cancer agents for the reduction of colorectal cancer.

Hydrolysis of glucosinolates using nylon-immobilized myrosinase to produce pure bioactive molecules.

Bioactive compounds were produced from natural glucosinolates, secondary plant metabolites, using myrosinase (thioglucoside glucohydrolase EC 3.2.3.1) isolated from ripe seeds of Sinapis alba. The enzyme was immobilized on granular nylon 6.6 with the crosslinking technique. Immobilized myrosinase displayed extraordinary operational and storage stability. Using a small thermostatted continuous packed-bed bioreactor, the enzyme activity was unchanged after 15 days of continuous use at 37 degrees C and after >1 year of storage at room temperature. The bioreactor was particularly efficient in producing pure isothiocyanates, but it was less efficient for pure nitrile production.

Modulation of GST P1-1 activity by polymerization during apoptosis.

Glutathione S-transferases (GSTs, EC 2.5.1.18) belong to a large family of functionally different enzymes that catalyze the S-conjugation of glutathione with a wide variety of electrophilic compounds including carcinogens and anticancer drugs. Drug resistance may result from reduction in apoptosis of neoplastic cells when exposed to antineoplastic drugs. The c-Jun N-terminal Kinase (JNK) belongs to the family of stress kinases and has been shown to be required for the maximal induction of apoptosis by DNA-damaging agents. Recently, an inhibition of JNK activity by GST P1-1, which was reversed by polymerization induced by oxidative stress, has been reported in 3T3-4A mouse fibroblast cell lines. The finding that GST P1-1 might inhibit JNK activity and that it is frequently highly expressed in tumor tissues suggests its possible implication in "apoptosis resistance" during antineoplastic therapy. We investigated the modulation of GST P1-1 during apoptosis in a neoplastic T-cell line (Jurkat) induced by hydrogen peroxide and etoposide. Apoptosis was paralleled by the appearance of a dimeric form of GST P1-1 on western blotting, associated with an increase in the Km(GSH) and a reduction in GST P1-1 specific activity toward 1-chloro-2,4-dinitrobenzene, which reached statistical significance only in H(2)O(2)-treated cells. Our data seem to suggest that H(2)O(2) and etoposide may partly act through a process of partial inactivation of the GST P1-1, possibly involving the "G" site in the process of dimerization, and thus favoring programmed cell death.

Formation of glucoraphanin by chemoselective oxidation of natural glucoerucin: a chemoenzymatic route to sulforaphane.

A new semi-synthetic way to produce glucoraphanin (2), the bio-precursor of the potential anticarcinogen sulforaphane (3), has been developed. Starting from glucoerucin (1), isolated from ripe seeds of Eruca sativa, glucoraphanin was obtained through chemoselective oxidation. Controlled myrosinase-catalysed hydrolysis of this precursor quantitatively afforded sulforaphane.

Myrosinase-generated isothiocyanate from glucosinolates: isolation, characterization and in vitro antiproliferative studies.

Epidemiological and pharmacological studies have shown that colorectal cancer development could be reduced by consuming vegetables that contain glucosinolates. In view of this the effect of some glucosinolates and their isothiocyanate (ITC)-derived products on in vitro cell growth was studied. We report the isolation and characterization of ITCs derived from glucosinolates by using HPLC, GC-MS, and NMR techniques. The in vitro activity of ITCs on human erythroleukemic K562 cells has been investigated by using two alternative approaches: the in situ and pre-mix methods. No differences in antiproliferative activity were found comparing the effect of ITCs produced either of these methods. In the experimental conditions used, the production of ITCs from glucosinolates is almost quantitative as confirmed by HPLC or GC-MS analysis. The ITCs' inhibitory activity on K562 cells growth is particularly evident in the cases of ITCs derived from sinigrin, progoitrin, epi-progoitrin, glucotropaeolin and glucocheirolin. Finally, the antiproliferative activity of the ITCs obtained from glucoraphenin, taken as an example, was determined on other tumor cell lines with a different origin and hystotype. Considering the antiproliferative activity found for ITCs these compounds could be considered potentially responsible for the reduction of colorectal cancer associated with diets rich in cruciferous vegetables. Further studies will be aimed at the possible application of glucosinolate-derived products as chemopreventive cancer agents.

The crystal structures of Sinapis alba myrosinase and a covalent glycosyl-enzyme intermediate provide insights into the substrate recognition and active-site machinery of an S-glycosidase.

BACKGROUND: Myrosinase is the enzyme responsible for the hydrolysis of a variety of plant anionic 1-thio-beta-D-glucosides called glucosinolates. Myrosinase and glucosinolates, which are stored in different tissues of the plant, are mixed during mastication generating toxic by-products that are believed to play a role in the plant defence system. Whilst O-glycosidases are extremely widespread in nature, myrosinase is the only known S-glycosidase. This intriguing enzyme, which shows sequence similarities with O-glycosidases, offers the opportunity to analyze the similarities and differences between enzymes hydrolyzing S- and O-glycosidic bonds. RESULTS: The structures of native myrosinase from white mustard seed (Sinapis alba) and of a stable glycosyl-enzyme intermediate have been solved at 1.6 A resolution. The protein folds into a (beta/alpha)8-barrel structure, very similar to that of the cyanogenic beta-glucosidase from white clover. The enzyme forms a dimer stabilized by a Zn2+ ion and is heavily glycosylated. At one glycosylation site the complete structure of a plant-specific heptasaccharide is observed. The myrosinase structure reveals a hydrophobic pocket, ideally situated for the binding of the hydrophobic sidechain of glucosinolates, and two arginine residues positioned for interaction with the sulphate group of the substrate. With the exception of the replacement of the general acid/base glutamate by a glutamine residue, the catalytic machinery of myrosinase is identical to that of the cyanogenic beta-glucosidase. The structure of the glycosyl-enzyme intermediate shows that the sugar ring is bound via an alpha-glycosidic linkage to Glu409, the catalytic nucleophile of myrosinase. CONCLUSIONS: The structure of myrosinase shows features which illustrate the adaptation of the plant enzyme to the dehydrated environment of the seed. The catalytic mechanism of myrosinase is explained by the excellent leaving group properties of the substrate aglycons, which do not require the assistance of an enzymatic acid catalyst. The replacement of the general acid/base glutamate of O-glycosidases by a glutamine residue in myrosinase suggests that for hydrolysis of the glycosyl-enzyme, the role of this residue is to ensure a precise positioning of a water molecule rather than to provide general base assistance.

A trypsin inhibitor from snail medic seeds active against pest proteases.

A protein trypsin inhibitor from seeds of snail medic (Medicago scutellata), MsTI, has been purified by ion-exchange chromatography, gel-filtration chromatography and reverse-phase HPLC. The protein inhibits the catalytic activity of bovine beta-trypsin, with an apparent Kd of 1.8 x 10(-9), but exhibits no activity towards bovine alpha-chymotrypsin. Moreover, MsTI inhibits the trypsin-like proteinase activity present in larvae of the crop pests Adoxophyes orana, Hyphantria cunea, Lobesia botrana and Ostrinia nubilalis. The complete amino acid sequence of MsTI consists of 62 residues corresponding to a M(r) of 6925. Sequence comparison shows that MsTI exhibits significant similarity to other proteins belonging to the Bowman-Birk trypsin inhibitor family, and the closest identity (81%) with the wound-induced trypsin inhibitor from Medicago sativa leaves.

Alpha but not beta interferon is useful in chronic active hepatitis due to hepatitis C virus. A prospective, double-blind, randomized study.

Interferon-alpha has been widely used in chronic hepatitis C, but controlled studies with intramuscular interferon-beta are lacking. We therefore performed a prospective, double-blind, randomized study comparing intramuscular IFN-alpha and -beta in patients with chronic hepatitis C. Sixty patients were randomly assigned to receive 3 MU thrice weekly intramuscularly of either recombinant IFN-alpha or leukocyte IFN-alpha or fibroblast IFN-beta for six months. Nine of 20 patients (45.0%) in the recombinant IFN, 5/19 (26.3%) in the leukocyte IFN, and none in the IFN-beta group had a complete response during therapy (recombinant IFN vs IFN-beta: P < 0.01). Only in IFN-alpha-treated patients, was infection with a single HCV genotype (type 2a or 2b) associated with significantly better long-term outcome. IFN-alpha is useful in chronic hepatitis C while intramuscular IFN-beta interferon does not exert any beneficial effect. This is probably due to an insufficient bioavailability of IFN-beta when given intramuscularly.

The myrosinase-glucosinolate interaction mechanism studied using some synthetic competitive inhibitors.

Using synthetic deoxy-glucotropaeolins (6d-GTL, 4d- GTL, 3d-GTL, 2d-GTL) as substrates, myrosinase activity was studied in comparison to that determined on native glucotropaeolin (GTL) isolated from ripe Lepidium sativum seeds. When the deoxy substrates were used, in addition to an overall strong reaction rate decline, a significant decrease in the reaction rate was observed in going from 6d- to 2d-GTL. This finding allows us to propose a mechanism of catalysis which appears to be similar in many respects to that established for beta-glucosidases. Finally, 2d-GTL was shown to be the first strong competitive inhibitor of myrosinase ever reported.

Determinants of bile secretion: effect of bile salt structure on bile flow and biliary cation secretion.

The effect of five bile salts, deoxycholate, chenodeoxycholate, cholate, ursodeoxycholate, and ursocholate, possessing (in decreasing order) different hydrophobicity, on bile flow and biliary secretion of total calcium, magnesium, sodium, and potassium was studied in 10 patients with T-tubes. Each subject was infused intraduodenally with one or two bile salts, given separately, to produce a selective enrichment of biliary bile salts with the infused bile salt. The choleresis induced per 1-mumol increase of bile salt output was greater during the secretion of 7 beta-hydroxylated bile salts, ursodeoxycholate (0.029 ml), and ursocholate (0.027 ml), followed in decreasing order by deoxycholate (0.023 ml), chenodeoxycholate (0.019 ml), and cholate (0.009 ml). Deoxycholate stimulated the greatest increase in cation secretion per unit increase in bile salt output, followed by chenodeoxycholate and cholate. The two 7 beta-hydroxylated bile salts induced greater cation secretion than did their 7 alpha-epimers. Whereas biliary concentration of divalent cations differed depending on the structure and concentration of the infused bile salt, the concentration of monovalent cations was constant for any species and concentration of infused bile salt. Relationships between bile salt and divalent cation concentration indicate that 1 mumol of secreted biliary deoxycholate, the most hydrophobic bile salt, associates with the greatest amount of calcium (0.046 mumol) and magnesium (0.022 mumol), followed by chenodeoxycholate (0.020 and 0.010 mumol, respectively) and cholate (0.012 and 0.008 mumol, respectively). The capacity of ursodeoxycholate and ursocholate to associate with calcium and magnesium seems to be less than that of their 7 alpha-epimers. These data suggest that of the common bile salts, the more hydrophobic bile salts stimulate bile flow and cation secretion better than the more hydrophilic bile salts, whereas ursodeoxycholate and ursocholate are more effective than their more hydrophobic 7 alpha-epimers. Whereas different bile salts seem to influence the secretion of sodium and potassium mainly by virtue of their choleretic properties, the effect of bile salt structure on biliary secretion of calcium and magnesium suggests the presence of a secretory link that might be consistent with cation-bile salt binding.