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PubMed1990

Modification of skeletal S-1 with fluorescein isothiocyanate.

1. Pyridoxal-5'-phosphate (PLP), a marker of primary amines, bound covalently to S-1 in an approximate ratio of 1:1. PLP was localized within the tryptic 25,000 mol. wt fragment. 2. Fluorescein isothiocyanate (FITC), which is known to bind covalently to primary amino groups located at nucleotide binding sites, readily bound to S-1 in a 2:1 ratio. FITC was localized within the 20,000 and 50,000 mol. wt tryptic peptides, in a ratio of 1:1 in each one. 3. These results are consistent with the existence of nucleotide binding sites on myosin different from those of the catalytic sites.

Adenosine Triphosphate↗
PubMed1988

Effect of allyl isothiocyanate on hepatic monooxygenases and serum transferases in rats.

The effect of allyl isothiocyanate (AITC) on the activities of liver monooxygenases and serum transferases was investigated in male rats. AITC was given in oral doses of 0, 50, 100 or 150 mg/kg bw for 3 consecutive days. Increased relative liver weights and decreased activities of hepatic aminopyrine-N-demethylase, p-nitroanisole-O-demethylase and aniline-p-hydroxylase were observed in all treatment groups. The administration of AITC at dose levels of 100 and 150 mg/kg caused a statistically significant increase in the serum aspartate aminotransferase activity.

Alanine Transaminase↗
PubMed1988

Sensitive method of detection, quantitation and purification of peptides using pre-column derivatization with phenyl isothiocyanate.

A sensitive method for the detection, quantitation and purification of peptides is described. The method is based on pre-column derivatization of peptides with phenyl isothiocyanate to form phenylthiocarbamoyl derivatives (PTC peptides). The derivatized peptides are analysed by reversed-phase high-performance liquid chromatography on a Zorbax ODS column (5 micron) and detected at 269 nm with a sensitivity limit of 1-5 pmol. The technique was utilized for the separation of a mixture of closely related synthetic peptides. The eluted PTC peptides were collected with an average recovery yield of 75% as determined by amino acid analysis. This method of separation of PTC peptides was also combined with the determination of the complete structure of recovered PTC-dynorphin A-(1-13) using the solid-phase sequenator (Sequemat). The advantages of the derivatization method are the rapidity and completeness of the reaction, the stability of the product, the sensitivity and specificity of the detection of derivatized peptides and the compatibility of the technique with subsequent analytical procedures. A particular application of this method was exemplified by the dosage of enkephalins secreted from perfused bovine adrenal glands.

Adrenal Medulla↗
PubMed2003

Determination of phenethyl isothiocyanate in human plasma and urine by ammonia derivatization and liquid chromatography-tandem mass spectrometry.

Phenethyl isothiocyanate (PEITC) is a dietary compound present in cruciferous vegetables that has cancer-preventive properties. Our objective was to develop and validate a novel liquid chromatography-tandem mass spectrometry procedure to analyze PEITC concentrations in human plasma and urine. Following hexane extraction, ammonia was added to samples to derivatize PEITC to phenethylthiourea. Chromatographic separation was achieved on a C(18) column with acetonitrile/5 mM formic acid (60:40, v/v) as the mobile phase followed by tandem mass spectrometry detection in multiple reaction monitoring mode. Deuterium-labeled PEITC was used as the internal standard. The detection limit was 2 nM and calibration curves were linear from 7.8 to 2000 nM. The intra- and inter-day coefficients of variation were less than 5 and 10%, respectively. The intra- and inter-day accuracies ranged from 101.0 to 104.2% and from 102.8 to 118.6%, respectively. The recovery from spiked human plasma and urine ranged from 100.3 to 113.5% and from 98.3 to 103.9%, respectively. The assay was used to measure PEITC in plasma and urine samples obtained from subjects after consumption of 100g of watercress. This novel assay represents the first analytical method with the sensitivity and specificity to determine plasma and urine concentrations of PEITC.

Adult↗
PubMed2004

Fluorescein isothiocyanate-hapten immunoassay for determination of peptide-cell interactions.

We have developed a fluorescein isothiocyanate (FITC)-hapten immunoassay, where a FITC-labeled peptide binding to a cell is assayed as the amount of immunoreactive fluorescein present in a cell lysate. An antifluorescein-horseradish peroxidase conjugate binds to either a fluoresceinated peptide in the lysate or a fluorescein attached to the wells of a microtiter plate in a competitive fashion. After washing, solid-phase peroxidase activity is measured and inversely related to the amount of FITC-labeled peptide present. To demonstrate the assay, the interaction of a FITC-labeled bombesin-like peptide with the gastrin-releasing peptide receptor on PC-3 and HT-29 cells was investigated. Using PC-3 cells, we obtained similar displacement curves and numbers of binding sites per cell by both the FITC-hapten immunoassay and a reference radioreceptor assay. The FITC-hapten immunoassay is a sensitive and versatile method, since the same commercially available reagents can be used to assess interactions between any peptide and any receptor. In addition, the FITC-labeled peptide can be used to visualize receptors in fluorescent-activated cell sorting or fluorescent microscopy.

Bombesin↗
PubMed2006

Sensitivity to allyl isothiocyanate, dimethyl trisulfide, sinigrin, and cooked cauliflower consumption.

The consumption of cauliflower consumers has been related to the olfactory and gustatory sensitivities to potentially objectionable flavor compounds in this vegetable. Based on the ascending concentration series method of limits, a first experiment was designed to develop rapid tests dedicated to estimate individuals' olfactory thresholds for allyl isothiocyanate (AITC) and dimethyl trisulfide (DMTS) and gustatory thresholds for sinigrin (SIN). The best compromise between rapidity and reliability was obtained with two replications of a four-alternative forced choice (AFC) at six ascending concentrations (6x2x4-AFC) for both AITC and DMTS, and with a 6x1x4-AFC for SIN. In a second experiment, sensitivity to SIN, AITC and DMTS was determined on 267 participants divided into three cauliflower consumer target groups: non-, medium- or high consumers. The non-consumers were significantly more sensitive to SIN and AITC than were the medium and high consumers. No effect of consumer's sensitivity to DMTS was observed.

Adolescent↗
PubMed2006

p38MAPK mediates benzyl isothiocyanate-induced p21WAF1 expression in vascular smooth muscle cells via the regulation of Sp1.

It has recently been reported that the transcription factors involved in p21WAF1 activation by certain signaling factors may vary in different cell types. However, the role and importance of the signaling pathway in the transcriptional regulation of p21WAF1 on vascular smooth muscle cells (VSMC) in response to benzyl isothiocyanate (BITC) has been unclear. In this report, we demonstrate that BITC induces the p21WAF1 expression at the transcriptional level. This increase in p21WAF1 gene expression was due to p38MAPK-dependent activation of the p21WAF1 promoter by BITC. Transcription factor Sp1 binding site was identified as the cis-element for the activation of p21WAF1 promoter by BITC, as determined by deletion and mutation analysis. In addition, gel shift and supershift assays demonstrated that this BITC-responsive element binds specifically to the transcription factor Sp1. Treatment with SB203580, an inhibitor of the p38MAPK, significantly downregulated transactivation of BITC-induced Sp1. Finally, the transient expression of VSMC with dominant negative p38MAPK plasmid suppressed BITC-stimulated Sp1 activity. In conclusion, we report that the transcription factor Sp1 involved in the p38MAPK-mediated control of p21WAF1 regulation on VSMC in response to BITC has now been identified.

Cells, Cultured↗
PubMed2005

Transport of dietary phenethyl isothiocyanate is mediated by multidrug resistance protein 2 but not P-glycoprotein.

We demonstrated recently that phenethyl isothiocyanate (PEITC), a potent anticarcinogen present in cruciferous vegetables, inhibited P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1) and that MRP1 can transport PEITC and/or its metabolites. In this study, we have examined whether PEITC is transported by P-gp and MRP2, two transporters with high expression in human intestine, liver and kidney. Using (14)C-PEITC, no significant difference was observed for the intracellular accumulation of PEITC in human breast cancer MCF-7/sensitive (control) and MCF-7/ADR (P-gp overexpressing) cells at PEITC concentrations of 1, 10 and 50 microM. Moreover, the presence of verapamil or PSC833, two P-gp inhibitors, had no significant effect on the intracellular accumulation of PEITC in P-gp overexpressing MCF-7/ADR and MDA435/LCC6MDR1 cells, indicating that PEITC may not be a substrate for P-gp. In contrast, (14)C-PEITC intracellular accumulation in the kidney epithelial MDCK II/MRP2 cells (transfected with human MRP2) was significantly lower than in the wild-type MDCK II/wt cells at PEITC concentrations of 1, 5, 10 and 50 microM. The presence of MK571, an MRP inhibitor, significantly enhanced (14)C-PEITC accumulation in MDCK II/MRP2 but not MDCK II/wt cells. Furthermore, depletion of intracellular glutathione (GSH) following treatment with buthionine sulphoximine, an inhibitor of GSH biosynthesis, significantly increased (14)C-PEITC intracellular accumulation in a concentration-dependent manner. Transcellular transport studies also demonstrated that depletion of intracellular GSH reduced the mean ratio of basal-to-apical transport to apical-to-basal transport of PEITC in MDCK II/MRP2, but not MDCK II/wt cell monolayers. These results indicate that GSH plays an important role in the MRP2-mediated transport of PEITC. The findings provide new information concerning the interactions between PEITC and membrane transporters and suggest the possibility of PEITC interactions with xenobiotics that are MRP2 substrates.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2005

Beta-phenylethyl isothiocyanate mediated apoptosis; contribution of Bax and the mitochondrial death pathway.

The initiating events that lead to the induction of apoptosis mediated by the chemopreventative agent beta-phenyethyl isothiocyanate (PEITC) have yet to be elucidated. In the present investigation, we examined the effects of PEITC on mitochondrial function and apoptotic signaling in hepatoma HepG2 cells and isolated rat hepatocyte mitochondria. PEITC induced a conformational change in Bax leading to its translocation to mitochondria in HepG2 cells. Bax accumulation was associated with a rapid loss of mitochondrial membrane potential (Deltapsim), impaired respiratory chain enzymatic activity, release of mitochondrial cytochrome c and the activation of caspase-dependent cell death. Caspase inhibition did not prevent Bax translocation, the release of cytochrome c or the loss of Deltapsim, but blocked caspase-mediated DNA fragmentation and cell death. To determine whether PEITC dependent Bax translocation caused loss of Deltapsim by the activation of the mitochondrial permeability transition (MPT), we examined the effects of PEITC in isolated rat hepatocyte mitochondria. Interestingly, PEITC did not induce MPT in isolated rat mitochondria. Accordingly, using pharmacological inhibitors of MPT namely cyclosporine A, trifluoperazine and Bongkrekic acid we were unable to block PEITC mediated apoptosis in HepG2 cells, this suggesting that mitochondrial permeablisation is a likely consequence of Bax dependent pore formation. Taken together, our data suggest that mitochondria are a key target in PEITC induced apoptosis in HepG2 cells via the pore forming ability of pro-apoptotic Bax.

Animals↗
PubMed2003

Phenethyl isothiocyanate inhibits nitrosamine carcinogenesis in a model for study of oral cancer chemoprevention.

The anticarcinogenic effect of phenethyl isothiocyanate (PEITC) was examined in hamster buccal pouch mucosa exposed to N-nitrosomethylbenzylamine (NMBA). Unscheduled DNA synthesis (UDS) and induction of intraepithelial gamma-glutamyl transpeptidase (gamma-GT) histochemical foci were assessed as potential predictors of anticarcinogenicity. UDS mediated by in vitro exposure to NMBA (at 10 and 50 mM) was examined in mucosal samples derived following topical exposure of pouch mucosa to PEITC at concentrations of 0.5, 5, or 50 mM. In vivo PEITC pretreatment reduced NMBA-induced UDS in a dose dependent manner. PEITC treatment reduced induction of gamma-GT foci, detected in epithelial wholemounts derived over a period of 8-13 weeks of NMBA application, by an average of 96%. PEITC also reduced tumor formation by 94%. gamma-GT, in particular, may be a useful indicator for identification of effective oral cancer chemopreventive agents and combinations of agents.

Animals↗
PubMed2004

Cellular accumulation of dietary anticarcinogenic isothiocyanates is followed by transporter-mediated export as dithiocarbamates.

Many dietary isothiocyanates (ITCs) are potent anticarcinogenic agents. ITCs rapidly accumulate to high concentrations in cells as a result of conjugation with intracellular thiols, especially glutathione (GSH). The anticarcinogenic activity of ITCs depends on, at least partly, their accumulation in cells. We report that three major anticarcinogenic ITCs, including allyl-ITC, benzyl-ITC, and phenethyl-ITC, were rapidly exported, upon accumulation in cells, mainly in the forms of GSH- and cysteinylglycine-conjugates, apparently involving MRP-1 and Pgp-1. These findings are consistent with our previous results regarding cellular export of another anticarcinogenic ITC, sulforaphane, and suggest a common cellular response to ITCs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Limited tumor-initiating activity of phenylethyl isothiocyanate by promotion with sodium L-ascorbate in a rat two-stage urinary bladder carcinogenesis model.

Initiation activity of phenylethyl isothiocyanate (PEITC) was examined in a two-stage urinary bladder carcinogenesis model. Male 6-week-old Fischer 344 rats were fed diet containing 0.1% PEITC for 12 or 24 weeks, with or without subsequent administration of 5% sodium l-ascorbate (Na-AsA) in diet until week 48, or for the entire experimental period. After 12 weeks of PEITC-treatment, both simple hyperplasia and papillary or nodular (PN) hyperplasia had developed in all animals, but the majority of these lesions had disappeared at week 48, irrespective of the Na-AsA-treatment. The same lesions after 24 weeks of PEITC-treatment had progressed to dysplasia and carcinoma, in a small number of cases by week 48 (6% in incidence for each lesion), but enhancement by the Na-AsA-treatment was evident only with simple hyperplasias (from 56 to 100% in incidence) and PN hyperplasias (from 19 to 56%). The results suggest a limited initiation activity of PEITC with induction of irreversible lesions by 24 weeks of exposure.

Animals↗
PubMed2005

The identification of 1,3-oxazolidine-2-thiones and 1,3-thiazolidine-2-thiones from the reaction of glucose with benzyl isothiocyanate.

The structure of interaction products resulting from the reaction of unmodified glucose with benzyl isothiocyanate is reported. Prior to their identification, the main products of this reaction were isolated using solid-phase extraction (SPE) as well as preparative HPLC. They were then identified by NMR and MS as 3-benzyl-4-hydroxy-5-(D-arabino-1,2,3,4-tetrahydroxybutyl)-1,3-oxazolidine-2-thione, 3-benzyl-4-hydroxy-4-hydroxymethyl-5-(D-erythro-1,2,3-trihydroxypropyl)-1,3-oxazolidine-2-thione, N-benzyl-(D-gluco-4,5-dihydroxy-6-hydroxymethyl-tetrahydropyrano)[2,3-b]oxazolidine-2-thione and 3-benzyl-4-(N-benzyl amino)-5-(D-arabino-1,2,3,4-tetrahydroxybutyl)-1,3-thiazolidine-2-thione. The identity of the last compound was secured by X-ray crystal structure data.

Chromatography, High Pressure Liquid↗
PubMed2006

Detection of hypoxia-related proteins in medaka (Oryzias latipes) brain tissue by difference gel electrophoresis and de novo sequencing of 4-sulfophenyl isothiocyanate-derivatized peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Two-dimensional fluorescence-based difference gel electrophoresis (DIGE) was used in combination with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) to identify a set of hypoxia-related biomarker proteins in medaka (Oryzias latipes) brain tissue. Each of the proteins were identified via de novo sequencing of tryptic peptides derivatized with 4-sulfophenyl isothiocyanate (SPITC), which N-terminally sulfonates peptides and promotes facile post-source decay peptide fragmentation, resulting in greatly simplified spectra consisting mainly of y-series fragment ions. We also report that addition of the non-ionic surfactant n-octyl-beta-d-glucopyranoside significantly improves SPITC-derivatized peptide recoveries. In addition, we found that a MALDI matrix consisting of the sodium-tolerant matrix 2,4,6-trihydroxyacetophenone, diammonium citrate, and alpha-cyano-4-hydroxycinnamic acid also improves ionization of SPITC-peptides, presumably by reducing ionization suppression effects from matrix contaminants, especially sodium cations. The DIGE experiments and analyses resulted in detection of six abundant proteins and related isozymes up-regulated (>1.49, p<0.005) in hypoxic medaka brain tissues, including two hemoglobin beta subunit forms, four carbonic anhydrase 2 forms, calbindin, aldolase, succinate dehydrogenase, and glutathione-S-transferase.

Animals↗
PubMed2006

Selective killing of oncogenically transformed cells through a ROS-mediated mechanism by beta-phenylethyl isothiocyanate.

Reactive oxygen species (ROS) stimulate cell proliferation and induce genetic instability, and their increase in cancer cells is often viewed as an adverse event. Here, we show that such abnormal increases in ROS can be exploited to selectively kill cancer cells using beta-phenylethyl isothiocyanate (PEITC). Oncogenic transformation of ovarian epithelial cells with H-Ras(V12) or expression of Bcr-Abl in hematopoietic cells causes elevated ROS generation and renders the malignant cells highly sensitive to PEITC, which effectively disables the glutathione antioxidant system and causes severe ROS accumulation preferentially in the transformed cells due to their active ROS output. Excessive ROS causes oxidative mitochondrial damage, inactivation of redox-sensitive molecules, and massive cell death. In vivo, PEITC exhibits therapeutic activity and prolongs animal survival.

Animals↗
PubMed2005

Selective sensitivity to wasabi-derived 6-(methylsulfinyl)hexyl isothiocyanate of human breast cancer and melanoma cell lines studied in vitro.

Recently, attention has focused on the anticancer properties of an aromatic component 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) in a typical Japanese spice, wasabi. In this paper, anticancer activity of 6-MITC in vitro was studied by using a human cancer cell (HCC) panel. 6-MITC directly affected the cells in the HCC panel and inhibited their growth in culture. The mean concentration required to inhibit 50% of control cell growth was 3.9 microM, which is a sufficiently low dosage for practical use. The suppression influenced not only the cell growth, but also the survival of these cells. The mean concentration to suppress cells to a 50% survival was 43.7 microM. The reduction activity of 6-MITC was differential, and it suppressed specific cells. These severely suppressed cell lines included breast cancer and melanoma cell lines. For example, one melanoma line was seriously damaged at a concentration of 0.3 microM of 6-MITC. Compared with other MITCs (2-MITC, 4-MITC and 8-MITC), 6-MITC showed the most effective suppression and with the most specific manner of the cells mentioned above. A "COMPARE" analysis using a computerized algorithm, which was based on the HCC database, suggested that the suppression mechanism of 6-MITC is unique and may be different from that of other known chemicals. The actual mechanism may not a simple one but may involve multiple pathways. On account of its sufficiently small size, 6-MITC is a new possible candidate for controlling cancer cells.

Algorithms↗
PubMed2006

Preventive effect of oral administration of 6-(methylsulfinyl)hexyl isothiocyanate derived from wasabi (Wasabia japonica Matsum) against pulmonary metastasis of B16-BL6 mouse melanoma cells.

AIM: Effect of oral administration of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) or a 6-MITC-containing T-wasabi fraction from wasabi root (Wasabia japonica Matsum) to inhibit the macroscopic pulmonary metastasis was studied with a murine B16-BL6 melanoma model. METHOD: Two administration routes, subcutaneous or intravenous, and two administration times, prior to or concomitant with tumor inoculation, of 6-MITC or T-wasabi against the metastatic foci formation in C57BL/6J mouse lungs were compared. RESULTS: The number of metastasized foci per lung in either subcutaneous or intravenous injection was significantly reduced by intake of 6-MITC or a T-wasabi fraction. The maximum reduction by a T-wasabi fraction reached to 82%. Fifty-six percent of foci formation was inhibited by a 2 week-prior administration of 6-MITC (200 microM), whereas only 27% inhibition was obtained by a concomitant administration with tumor inoculation. Neither 6-MITC nor T-wasabi at tested concentrations showed any toxic effects. DISCUSSION: Together with our previous results, a component of the Japanese pungent spice, wasabi appears to inhibit not only tumor cell growth but also tumor metastasis. Therefore, 6-MITC from wasabi is apparently a useful dietary candidate for controlling tumor progression.

Administration, Oral↗
PubMed2004

Monitoring of the conjugation reaction between human serum transferrin and fluorescein isothiocyanate by capillary electrophoresis.

Labeling of iron-free human serum transferrin by an amine-reactive probe, fluorescein isothiocyanate (FITC) was monitored with different dye to protein ratios. The degree of labeling was followed and determined by capillary electrophoresis. Depending on the number of the bound FITC, a shift in the electropherogram was observed, but the conjugated-transferrin forms were resolved from the unbound FITC in zone electrophoresis. The lowest protein concentration that resulted in detectable transferrin conjugate was 0.13 microM, and the limit of detection of the conjugated protein was 0.13 nM (10 ng/ml) with a signal to noise ratio of one to three.

Electrophoresis, Capillary↗