Isothiocyanate Content Research Articles

Effects of changes in pH and temperature on the inhibition of Salmonella and Listeria monocytogenes by Allyl isothiocyanate

The minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) of mustard allyl isothiocyanate (AITC) against five strains each of Salmonella and Listeria monocytogenes individually, and when combined by genus were studied in Mueller-Hinton broth at 21 or 37 °C as well as the interactive effects of pH (5.0–9.0) at temperatures of 4–21 °C on cell viability when held under these conditions for up to 10 d. The stability of 200 ppm AITC was monitored during these trials. The MIC and MBC values of AITC ranged from 60 to 100 ppm and 120–180 ppm, respectively, at 37 °C and ranged from 10 to 40 ppm and 200–600 ppm, respectively, at 21 °C against both pathogens. AITC had no antimicrobial activity at low temperatures (4 or 10 °C) and alkaline pH over 10 d, but at neutral pH, AITC reduced L. monocytogenes by 4.14 and 8.45 log10 CFU/ml at 4 or 10 °C, respectively. At acidic pH, AITC was more effective against Salmonella which was reduced by 2.56 and 6.48 log10 CFU/ml at 4 and 10 °C, respectively. However, AITC was more effective at combinations of 21 °C and neutral pH against L. monocytogenes (cells were not detected at and beyond 3 d) and at combinations of the higher temperature and acidic pH against Salmonella (cells were not detected at and beyond 6 d). Mustard AITC was more stable at low pH and temperature indicating that it can be an effective antimicrobial at combinations of low or neutral pH values and room or refrigerator temperatures (4–10 °C) against these foodborne pathogens.

The Isothiocyanate, Sulforaphane, Alters the Acidification of the Vacuole to Trigger Yeast Cell Death

The isothiocyanate, sulforaphane (SFN), isolated from cruciferous vegetables is a potential chemotherapeutic agent. Recent studies in mammalian cells have suggested that SFN works by causing cell cycle arrest and/or initiating programmed cell death (PCD). In order to identify genes that may be involved in the eukaryotic cell's response to SFN, we initiated a genetic screen using the yeast knockout (YKO) library of the budding yeast, Saccharomyces cerevisiae, to identify loss of function mutants that are hypersensitive to SFN. We have identified 311 SFN‐sensitive and 11 SFN‐resistant mutants in yeast ORFs that had been previously linked to vacuolar acidification suggesting that SFN kills cells by altering the acidification of the yeast vacuole. Staining with BCECF‐AM confirmed that SFN makes yeast vacuoles more basic. Strikingly, cells cultured in lethal concentrations of benzyl isothiocyanate and phenethyl isothiocyanate did not exhibit changes in vacuole acidification, suggesting that this trigger of programmed cell death is unique to sulforaphane. [Our laboratory is supported by the following grants: NIGMS R15 GM094712, NSF MRI‐R2 0959354 and NIH Grant 2 P20 RR016457 to the Rhode Island INBRE Program]

Corn oil and milk enhance the absorption of orally administered allyl isothiocyanate in rats

Allyl isothiocyanate, a chief component of mustard oil, exhibits anticancer effects in both cultured cancer cells and animal models. The accumulation of the N-acetylcysteine conjugate of allyl isothiocyanate, the final metabolite of allyl isothiocyanate, in urine was evaluated in rats that were orally coadministered allyl isothiocyanate with fluids (e.g., water, green tea, milk, and 10% ethanol) or corn oil. The N-acetylcysteine conjugate of allyl isothiocyanate content in urine when allyl isothiocyanate (2 or 4μmol) was coadministered with corn oil or milk showed a greater increase (1.4±0.22 or 2.7±0.34μmol or 1.2±0.32 or 2.5±0.36μmol, 1.6- to 1.8-fold or 1.5-fold, respectively) than when allyl isothiocyanate (2 or 4μmol) was coadministered with water (0.78±0.10 or 1.7±0.17μmol). This result demonstrates that corn oil and milk enhance the absorption of allyl isothiocyanate in rats.

Comparison of bioactive phytochemical content and release of isothiocyanates in selected brassica sprouts

The consumption of brassica sprouts as raw vegetables provides a fair amount of glucosinolates (GLs) and active plant myrosinase, which enables the breakdown of GLs into health-promoting isothiocyanates (ITCs). This study reports the determination of the main constituents related to human health found in edible sprouts of two Brassica oleracea varieties, broccoli and Tuscan black kale, and two Raphanus sativus varieties, Daikon and Sango. Radish sprouts exhibited the highest ability to produce ITCs, with Daikon showing the greatest level of conversion of GLs into bioactive ITCs (96.5%), followed by Sango (90.0%). Tuscan black kale gave a value of 68.5%, whereas broccoli displayed the lowest with 18.7%. ITCs were not the exclusive GL breakdown products in the two B. oleracea varieties, since nitriles were also produced, thus accounting for the lower conversion observed. Measuring the release of plant ITCs is a valuable tool in predicting the potential level of exposure to these bioactive compounds after the consumption of raw brassica sprouts.

Inter simple sequence repeat polymorphism in Alternaria genomic DNA exposed to lethal concentrations of isothiocyanates

Isothiocyanates (ITCs) is a group of defense related compounds synthesized byBrassicas that have positive effects on human health which suggest that they are environmentally friendly compounds to control fungi infections. However, the development of ITC-resistant strains of fungi is a matter of concern. The objective of the present work was to study the response of Alternaria alternata to allyl-isothiocyanate (AITC) and benzyl isothiocyanate (BITC) chronic exposure and the effect of the treatment on some of the inter simple sequence repeats (ISSR) regions. Five strains of A. alternata isolated from tomato and five isolated from cabbage were independently exposed in vitro to AITC and BITC. Concentrations were increased until it reached 0.08 and 0.6 mg/mL of AITC and BITC, respectively. Genomic DNA from both wild type and isogenic adapted strains to ITCs was isolated and five primers were used for ISSR amplification: (GACA)4: 5´-GACAGACAGACAGACA-3´; M13:5´-GAGGGTGGCGGCGGTTCT-3´;(AAG)8:5´-AAGAAGAAGAAGAAGAAGAAGAAG-3´; (ACA)5: 5´-ACAACAACAACAACA-3´ and T3B: 5´-AGGTCGCGGGTTCGAATCC-3´. A lower degree of polymorphism was found to be induced by the treatment in the wild-type strains isolated from cabbage as compared with the wild-type strains isolated from tomato. It can be concluded that ITCs exposure induced random mutations in different ISSR regions of the A. alternata genome which does not lead to the development of strains with a hereditable resistant phenotype. Key words: Alternaria alternata, fungicide, allyl-isothiocyanate, benzyl-isothiocyanate, inter-simple sequence repeat

Assessing the Impact of High-Pressure Processing on Selected Physical and Biochemical Attributes of White Cabbage (Brassica oleracea L. var. capitata alba)

The aim of this study was to investigate the effect of combined pressure/temperature treatments (200, 400 and 600 MPa, at 20 and 40 °C) on key physical and chemical characteristics of white cabbage (Brassica oleracea L. var. capitata alba). Thermal treatment (blanching) was also investigated and compared with high-pressure processing (HPP). HPP at 400 MPa and 20–40 °C caused significantly larger colour changes compared to any other pressure or thermal treatment. All pressure treatments induced a softening effect, whereas blanching did not significantly alter texture. Both blanching and pressure treatments resulted in a reduction in the levels of ascorbic acid, effect that was less pronounced for blanching and HPP at 600 MPa and 20–40 °C. HPP at 600 MPa resulted in significantly higher total phenol content, total antioxidant capacity and total isothiocyanate content compared to blanching. In summary, the colour and texture of white cabbage were better preserved by blanching. However, HPP at 600 MPa resulted in significantly higher levels of phytochemical compounds. The results of this study suggest that HPP may represent an attractive technology to process vegetable-based food products that better maintains important aspects related to the content of health-promoting compounds. This may be of particular relevance to the food industry sector involved in the development of convenient novel food products with excellent functional properties.

Pungency of Red Radish Storage Roots Is Unaffected by Spent Mushroom Substrate or Yard Waste Compost Treatments Used for Field Production

Field experiments were conducted at Bixby, OK, in 2007. Four compost treatments and an unamended control were compared for field production of eight (spring) or four (fall) red radish (Raphanus sativus L.) cultivars. Treatments were either spent mushroom substrate or yard waste compost spread over plots to an average depth of 2.5 or 5 cm and preplant-incorporated ≈5 to 7 cm deep. Radishes were direct-seeded into prepared plots and subsequently grown using standard cultural practices. Samples of median-sized marketable storage roots were shredded and juice was analyzed in the laboratory for pungency as measured by isothiocyanate (ITC) concentration (primarily 4-methylthio-3-butenyl isothiocyanate). In the spring, mean ITC concentrations ranged from 28.2 to 36.8 μmol per 100 g juice in storage roots from the four compost treatments, and differences were not significant (α = 0.05). There were not enough storage roots to analyze from the unamended control plots as a result of herbicide toxicity. Cultivars differed in mean concentration of ITCs, ranging from a high of 52.9 μmol per 100 g juice for ‘Cherry Belle’ to a low of 19.2 μmol per 100 g juice for ‘Crunchy Royale’. In the fall, mean ITC concentrations ranged from 10.5 to 24.6 μmol per 100 g juice in storage roots from the four compost treatments. Differences were not significant (α = 0.05), and there were no differences from the control value of 17.5 μmol per 100 g juice. The mean ITC concentration was 19.9 μmol per 100 g juice for the four cultivars tested in the fall, and the cultivars did not differ. Results indicate that the tested compost treatments did not affect pungency of red radish storage roots as measured by concentrations of ITCs.

Increased pepper yields following incorporation of biofumigation cover crops and the effects on soilborne pathogen populations and pepper diseases

The use of brassica cover crops and their associated degradation compounds as biofumigants to manage soilborne pathogens could offer vegetable growers an alternative to the restricted broad-spectrum fumigant methyl bromide. Biofumigation was tested in two experiments to manage Rhizoctonia solani, Sclerotium rolfsii, and Pythium spp., and the diseases they cause on pepper. Field plots were seeded in fall 2010 and spring 2011 to oilseed radish (Raphanus sativus L.), ‘Pacific Gold’ mustard (Brassica juncea (L.) Czern), or ‘Dwarf Essex’ winter rapeseed (Brassica napus L.). Cover crops were disked into soil in spring 2011 and immediately covered with virtually impermeable film (VIF) to reduce the escape of volatile pesticidal compounds. Controls included fallow plots with (CVIF) and without (fallow) VIF. Green bell pepper was transplanted into all plots. Concentrations of isothiocyanates (ITCs), the brassica degradation compounds primarily responsible for pesticidal activity, were highest following incorporation of mustard. Rapeseed yielded the second highest ITC concentrations. Radish yielded very low ITC concentrations in experiment 1, and none during experiment 2. ITCs also were detected in low concentrations in CVIF treatments. All treatments that received VIF reduced populations of R. solani compared to fallow, with no differences between biofumigation treatments and CVIF. Biofumigation treatments did not reduce populations of Pythium spp. or S. rolfsii compared to CVIF. Pepper stunting was significantly lower in treatments that received VIF compared to fallow, with no consistently significant differences between biofumigation treatments and CVIF. Pythium isolated from roots of stunted peppers was identified as Pythium aphanidermatum. Biofumigation treatments did not reduce plant mortality. Pepper yields were highest in biofumigation treatments compared to CVIF, and CVIF yields were higher than fallow yields.

Comparison of the Herbicidal Activity of Phenyl Isothiocyanate with Methyl Bromide in Polyethylene-Mulched Tomato

Field experiments were conducted in 2006 and 2007 to evaluate the herbicidal activity of phenyl isothiocyanate (ITC) on yellow nutsedge, Palmer amaranth, and large crabgrass in tomato grown on two polyethylene-mulched types. Treatments included two mulch types (low density polyethylene [LDPE] mulch and virtually impermeable film [VIF] mulch) and phenyl ITC at 0, 15, 75, 150, 750, and 1,500 kg ha−1. A standard rate of methyl bromide/chloropicrin (67 : 33%) at 390 kg ha−1under LDPE mulch was included for comparison. Regardless of mulch type, phenyl ITC at 1,452 (±133) and 1,719 (±426) kg ha−1was required for broad-spectrum weed control equivalent to methyl bromide in 2006 and 2007, respectively. Tomato injury was ≥ 44% at the highest phenyl ITC rate of 1,500 kg ha−1at 2 wk after transplanting (WATP) both years, irrespective of mulch type. Greater crop injury was observed from 750 kg ha−1of phenyl ITC in 2006 (≥ 27%) than in 2007 (≤ 10%). The greater injury in 2006 was attributed to a higher phenyl ITC concentration because holes in the plastic mulch for transplanting were punched at the time of transplanting in 2006; whereas, in 2007 holes were punched 2 d before transplanting, allowing 2 d of aeration before transplanting. Tomato marketable yield at all rates of phenyl ITC was lower than with methyl bromide in 2006. However, in 2007, marketable yield in plots treated with phenyl ITC at 750 kg ha−1was equivalent to methyl bromide. Overall, VIF mulch was no more effective than LDPE mulch at increasing weed control or improving the marketable yield of tomato either year.

Isothiocyanate concentrations and interconversion of sulforaphane to erucin in human subjects after consumption of commercial frozen broccoli compared to fresh broccoli

Sulforaphane (a potent anticarcinogenic isothiocyanate derived from glucoraphanin) is widely considered responsible for the protective effects of broccoli consumption. Broccoli is typically purchased fresh or frozen and cooked before consumption. We compared the bioavailability and metabolism of sulforaphane from portions of lightly cooked fresh or frozen broccoli, and investigated the bioconversion of sulforaphane to erucin. Eighteen healthy volunteers consumed broccoli soups produced from fresh or frozen broccoli florets that had been lightly cooked and sulforaphane thio-conjugates quantified in plasma and urine. Sulforaphane bioavailability was about tenfold higher for the soups made from fresh compared to frozen broccoli, and the reduction was shown to be due to destruction of myrosinase activity by the commercial blanching-freezing process. Sulforaphane appeared in plasma and urine in its free form and as several thio-conjugates forms. Erucin N-acetyl-cysteine conjugate was a significant urinary metabolite, and it was shown that human gut microflora can produce sulforaphane, erucin, and their nitriles from glucoraphanin. The short period of blanching used to produce commercial frozen broccoli destroys myrosinase and substantially reduces sulforaphane bioavailability. Sulforaphane was converted to erucin and excreted in urine, and it was shown that human colonic flora were capable of this conversion.

In vitro efficacies of various isothiocyanates from cruciferous vegetables as antimicrobial agents against foodborne pathogens and spoilage bacteria

The antimicrobial activities of ten isothiocyanates (ITCs), namely sulforaphane, iberin, allyl-, benzyl-, methyl-, phenyl-, phenylethyl-, propyl-, 3-methylthiophenyl-, and 3-methylthiopropyl-ITC, were tested against a range of pathogenic and food spoilage bacteria. The activities of six ITCs were investigated for the first time. The growth of the bacteria was monitored by turbidimetry in a culture broth with increasing concentrations of ITCs. An antimicrobial efficacy index (IAE) was calculated based on the observed growth delay, reduction in maximum growth rate and reduction in population size. ITCs inhibited pathogenic and food spoilage bacteria including species from Bacillus, Escherichia, Klebsiella, Listeria, Salmonella, Serratia and Staphylococcus. Gram-negative bacteria were overall more sensitive to ITCs than their Gram-positive counterparts: the three most sensitive species with five ITCs, and two out of three most sensitive species with the other ITCs. Yet, the species of a bacterium seemed to play a more important role, since considerable variations in sensitivity were observed between species even within the same Gram type. All ITCs investigated displayed antimicrobial activity, but with levels that depended on the target bacteria and the molecule considered. The highest IAE values were obtained with Benzyl-ITC, followed by phenylethyl-ITC, but this was not necessarily the case with all aromatic ITCs. An aliphatic compound, 3-methylthiopropyl-ITC, was much more active than phenyl-ITC and 3-methylthiophenyl-ITC. Within a structural group, the activity of ITCs varied dramatically, and structural features, like the presence of a sulfinyl group, the molecule size, and the length of the hydrocarbon chain, seem to be of importance.

Low concentrations of isothiocyanates protect mesenchymal stem cells from oxidative injuries, while high concentrations exacerbate DNA damage

Isothiocyanates (ITCs) are molecules naturally present in many cruciferous vegetables (broccoli, black radish, daikon radish, and cauliflowers). Several studies suggest that cruciferous vegetable consumption may reduce cancer risk and slow the aging process. To investigate the effect of ITCs on cellular DNA damage, we evaluated the effects of two different ITCs [sulforaphane (SFN) and raphasatin (RPS)] on the biology of human mesenchymal stem cells (MSCs), which, in addition to their ability to differentiate into mesenchymal tissues, contribute to the homeostatic maintenance of many organs. The choice of SFN and RPS relies on two considerations: they are among the most popular cruciferous vegetables in the diet of western and eastern countries, respectively, and their bioactive properties may differ since they possess specific molecular moiety. Our investigation evidenced that MSCs incubated with low doses of SFN and RPS show reduced in vitro oxidative stress. Moreover, these cells are protected from oxidative damages induced by hydrogen peroxide, while no protection was evident following treatment with the UV ray of a double strand DNA damaging drug, such as doxorubicin. High concentrations of both ITCs induced cytotoxic effects in MSC cultures and further increased DNA damage induced by peroxides. In summary, our study suggests that ITCs, at low doses, may contribute to slowing the aging process related to oxidative DNA damage. Moreover, in cancer treatment, low doses of ITCs may be used as an adjuvant to reduce chemotherapy-induced oxidative stress, while high doses may synergize with anticancer drugs to promote cell DNA damage.

Comparative study between extraction techniques and column separation for the quantification of sinigrin and total isothiocyanates in mustard seed

Glucosinolates are β-thioglycosides which are found naturally in Cruciferae including the genus Brassica. When enzymatically hydrolysed, glucosinolates yield isothiocyanates and give a pungent taste. Both glucosinolates and isothiocyanates have been linked with anticancer activity as well as antifungal and antibacterial properties and therefore the quantification of these compounds is scientifically important. A wide range of literature exists on glucosinolates, however the extraction and quantification procedures differ greatly resulting in discrepancies between studies. The aim of this study was therefore to compare the most popular extraction procedures to identify the most efficacious method and whether each extraction can also be used for the quantification of total isothiocyanates. Four extraction techniques were compared for the quantification of sinigrin from mustard cv. Centennial (Brassica juncea L.) seed; boiling water, boiling 50% (v/v) aqueous acetonitrile, boiling 100% methanol and 70% (v/v) aqueous methanol at 70°C. Prior to injection into the HPLC, the extractions which involved solvents (acetonitrile or methanol) were freeze-dried and resuspended in water. To identify whether the same extract could be used to measure total isothiocyanates, a dichloromethane extraction was carried out on the sinigrin extracts. For the quantification of sinigrin alone, boiling 50% (v/v) acetonitrile was found to be the most efficacious extraction solvent of the four tested yielding 15% more sinigrin than the water extraction. However, the removal of the acetonitrile by freeze-drying had a negative impact on the isothiocyanate content. Quantification of both sinigrin and total isothiocyanates was possible when the sinigrin was extracted using boiling water. Two columns were compared for the quantification of sinigrin revealing the Zorbax Eclipse to be the best column using this particular method.

Abstract 5430: Analysis of genome-wide DNA methylation in CCRF-CEM cells confirms hypermethylation at reported epigenetic markers of T-cell lymphoblastic leukemia and identifies novel therapeutic targets with reduced methylation upon treatment with dietary indoles

Abstract Acute lymphoblastic leukemia/lymphoma (ALL) is the most common class of malignancies among children and cases of T-cell origin are less responsive to therapy than their B-cell counterparts. Cruciferous vegetables have been demonstrated to be chemoprotective in a number of cancer cell types due to their high content of indoles and isothiocyanates. Previously, we demonstrated that indole-3-carbinol (I3C) is an effective transplacental chemopreventive agent in a dibenzo[def, p]chrysene (DBC)-induced model of murine T-ALL. We further confirmed that treatment of human T-ALL cell lines with I3C and diindolylmethane (DIM), the primary product of I3C condensation, reduces cell proliferation, viability, and expression of cell-cycle proteins. Aberrant DNA methylation is a recognized event in the development of cancer and pharmacological demethylation of promoter-associated CpG islands is of therapeutic interest to restore expression of epigenetically silenced genes. We utilized genome-wide DNA methylation analysis to identify potential epigenetic therapeutic targets of I3C and DIM in a model of T-ALL. By differential methylation region (DMR) analysis with stringent bonferroni multiple testing correction, 1,699 DMR probes were identified which mapped to 298 unique transcripts. The differential degree of hypomethylation by I3C, compared with DIM, is consistent with previous findings that DIM is a more potent therapeutic in this model. I3C (60 µM) significantly reduced methylation in 53% of DMRs yet increased methylation in the remaining 173 gene-mapped DMRs. DIM treatment with the consumer available supplement formulation known as Bioresponse-DIM (BR-DIM; 5 µM), or with the pure crystalline form (C-DIM; 5 µM or 15 µM), reduced methylation in over 500 probes and more than 200 genes. Functional annotation of differentially hypomethylated genes revealed that homeobox genes and transcription factors were most commonly represented with negative regulators of apoptosis and Wnt/Frizzled families also among the top gene clusters. Many identified genes have been previously recognized as being frequently hypermethylated in hematological malignancies and our results support demethylation of these critical cancer survival pathways by dietary indoles derived from cruciferous vegetables. These and novel genes from our analysis are currently being validated and examined at the level of transcription. Supported by P01CA90890, P42ES016465, CA135523 and The Linus Pauling Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5430. doi:1538-7445.AM2012-5430

Insecticidal fumigant action of mustard essential oil against Sitophilus zeamais in maize grains

This study was done to evaluate the fumigant toxicity of synthetic mustard essential oil (SMEO) (90% allyl isothiocyanate) on the developmental stages of the maize weevil (Sitophilus zeamais) and its effect on maize grain quality. Time-response bioassays were carried out in maize grains infested with eggs, 3rd instar larvae, pupae, or adults of the pest, using SMEO to provide allyl isothiocyanate (AITC) concentrations of 1.25 or 1.87 μl l−1, for 60 h with assessment after an interval of 6 h or until 100% mortality was reached. The vapors of SMEO caused mortality of all stages of S. zeamais. However, eggs were significantly more tolerant than the other developmental stages, followed by pupae. The larvae were least tolerant. The moisture content, electric conductivity and germination of the grains was not affected by exposure to SMEO. This exploratory study shows that SMEO can be a good candidate for development as an eco-friendly grain fumigant for weevil control.

Natural isothiocyanates: Genotoxic potential versus chemoprevention

Isothiocyanates, occurring in many dietary cruciferous vegetables, show interesting chemopreventive activities against several chronic-degenerative diseases, including cancer, cardiovascular diseases, neurodegeneration, diabetes. The electrophilic carbon residue in the isothiocyanate moiety reacts with biological nucleophiles and modification of proteins is recognized as a key mechanism underlying the biological activity of isothiocyanates. The nuclear factor-erythroid-2-related factor 2 system, which orchestrates the expression of a wide array of antioxidant genes, plays a role in the protective effect of isothiocyanates against almost all the pathological conditions reported above. Recent emerging findings suggest a further common mechanism. Chronic inflammation plays a central role in many human diseases and isothiocyanates inhibit the activity of many inflammation components, suppress cyclooxygenase 2, and irreversibly inactivate the macrophage migration inhibitory factor. Due to their electrophilic reactivity, some isothiocyanates are able to form adducts with DNA and induce gene mutations and chromosomal aberrations. DNA damage has been demonstrated to be involved in the pathogenesis of various chronic-degenerative diseases of epidemiological relevance. Thus, the genotoxicity of the isothiocyanates should be carefully considered. In addition, the dose-response relationship for genotoxic compounds does not suggest evidence of a threshold. Thus, chemicals that are genotoxic pose a greater potential risk to humans than non-genotoxic compounds. Dietary consumption levels of isothiocyanates appear to be several orders of magnitude lower than the doses used in the genotoxicity studies and thus it is highly unlikely that such toxicities would occur in humans. However, the beneficial properties of isothiocyanates stimulated an increase of dietary supplements and functional foods with highly enriched isothiocyanate concentrations on the market. Whether such concentrations may exert a potential health risk cannot be excluded with certainty and an accurate evaluation of the toxicological profile of isothiocyanates should be prompted before any major increase in their consumption be recommended or their clinical use suggested.

Carrageenans, sulphated polysaccharides of red seaweeds, differentially affect Arabidopsis thaliana resistance to Trichoplusia ni (cabbage looper).

Carrageenans are a collective family of linear, sulphated galactans found in a number of commercially important species of marine red alga. These polysaccharides are known to elicit defense responses in plant and animals and possess anti-viral properties. We investigated the effect of foliar application of ι-, κ- and λ-carrageenans (representing various levels of sulphation) on Arabidopsis thaliana in resistance to the generalist insect Trichoplusia ni (cabbage looper) which is known to cause serious economic losses in crop plants. Plants treated with ι- and κ-carrageenan showed reduced leaf damage, whereas those treated with λ- carrageenan were similar to that of the control. In a no-choice test, larval weight was reduced by more than 20% in ι- and κ- carrageenan treatments, but unaffected by λ-carrageenan. In multiple choice tests, carrageenan treated plants attracted fewer T. ni larvae by the fourth day following infestation as compared to the control. The application of carrageenans did not affect oviposition behaviour of T. ni. Growth of T. ni feeding on an artificial diet amended with carrageenans was not different from that fed with untreated control diet. ι-carrageenan induced the expression of defense genes; PR1, PDF1.2, and TI1, but κ- and λ-carrageenans did not. Besides PR1, PDF1.2, and TI1, the indole glucosinolate biosynthesis genes CYP79B2, CYP83B1 and glucosinolate hydrolysing QTL, ESM1 were up-regulated by ι-carrageenan treatment at 48 h post infestation. Gas chromatography-mass spectrometry analysis of carrageenan treated leaves showed increased concentrations of both isothiocyanates and nitriles. Taken together, these results show that carrageenans have differential effects on Arabidopsis resistance to T. ni and that the degree of sulphation of the polysaccharide chain may well mediate this effect.

Contents of sulforaphane and total isothiocyanates, antimutagenic activity, and inhibition of clastogenicity in pulp

The consumption of Cruciferous vegetables is important for the prevention of cancerous diseases, particularly colorectal cancer. The effects of technological treatments (freezing, pasteurisation, high-pressure treatment) on the content of isothiocyanates, considered to be the active substance, were observed in single-species vegetable juices prepared from cruciferous vegetables (broccoli, cauliflower, Brussels sprouts, white and red cabbage). The contents of sulforaphane and total isothiocyanates were studied relative to the temperature, action period, and time delay after juice pressing. Sulforaphane and total isothiocyanates were determined by HPLC. Sulforaphane content in various parts of fresh broccoli was also assessed. Antimutagenic activity of the juices (frozen, pasteurised, and high-pressure treated) was evaluated using the Ames test and the following mutagens:  AFTB1 (aflatoxin B1), IQ (2-amino-3-methyl-3H-imidazo-[4,5-f]quinoline), and MNU (2-nitroso-2-methylurea). Clastogenicity inhibition of the mutagens, in response to broccoli juice, as well as of pure sulforaphane, was observed using an in vivo experiment (the micronucleus test). It was shown that in terms of sulforaphane content, it is best to let broccoli juice stand for 60 min after pressing and pH adjustment. Sulforaphane content does not change under heating to 60°C. Its content decreases considerably (compared to fresh juice) with heating to higher temperatures than 60°C. High-pressure treatment preserves mutagenic inhibition to the same degree as juices freezing.

Activation characteristics of transient receptor potential ankyrin 1 and its role in nociception

Transient receptor potential (TRP) ankyrin 1 (TRPA1) is a Ca(2+)-permeant, nonselective cationic channel. It is predominantly expressed in the C afferent sensory nerve fibers of trigeminal and dorsal root ganglion neurons and is highly coexpressed with the nociceptive ion channel transient receptor potential vanilloid 1 (TRPV1). Several physical and chemical stimuli have been shown to activate the channel. In this study, we have used electrophysiological techniques and behavioral models to characterize the properties of TRPA1. Whole cell TRPA1 currents induced by brief application of lower concentrations of N-methyl maleimide (NMM) or allyl isothiocyanate (AITC) can be reversed readily by washout, whereas continuous application of higher concentrations of NMM or AITC completely desensitized the currents. The deactivation and desensitization kinetics differed between NMM and AITC. TRPA1 current amplitude increased with repeated application of lower concentrations of AITC, whereas saturating concentrations of AITC induced tachyphylaxis, which was more pronounced in the presence of extracellular Ca(2+). The outward rectification exhibited by native TRPA1-mediated whole cell and single-channel currents was minimal as compared with other TRP channels. TRPA1 currents were negatively modulated by protons and polyamines, both of which activate the heat-sensitive channel, TRPV1. Interestingly, neither protein kinase C nor protein kinase A activation sensitized AITC-induced currents, but each profoundly sensitized capsaicin-induced currents. Current-clamp experiments revealed that AITC produced a slow and sustained depolarization as compared with capsaicin. TRPA1 is also expressed at the central terminals of nociceptors at the caudal spinal trigeminal nucleus. Activation of TRPA1 in this area increases the frequency and amplitude of miniature excitatory or inhibitory postsynaptic currents. In behavioral studies, intraplantar and intrathecal administration of AITC induced more pronounced and prolonged changes in nociceptive behavior than those induced by capsaicin. In conclusion, the characteristics of TRPA1 we have delineated suggest that it might play a unique role in nociception.

Glucosinolate and Isothiocyanate Production from Brassicaceae Cover Crops in a Plasticulture Production System

Brassicaceae cover crops are gaining attention as potential biofumigants for soil pest suppression because of their ability to release biologically active isothiocyanates (ITCs) and other compounds from hydrolysis of glucosinolates (GSLs). However, biofumigation potential of a Brassicaceae is related to its GSL and ITC profile and GSL to ITC conversion efficiency. Field and laboratory experiments were conducted to evaluate the biofumigation potential of seven Brassicaceae cover crops for weed control in plasticulture tomato and bell pepper. GSL concentration and composition varied among cover crops and between roots and shoots of each cover crop. Similar GSLs were produced in both years by roots or shoots of each cover crop, but GSL concentrations were variable between years. Total GSLs contributed to the soil by incorporation of Brassicaceae cover crop tissues were estimated between 47 to 452 nmol g−1soil. Highest ITC concentration was detected in soil at 3 h after cover crop incorporation, and concentration decreased at later timings. GSL to ITC conversion efficiency ranged from 1 to 39%, with variation among cover crops and between years. No injury was observed in tomato and bell pepper transplanted 1 wk after cover crop incorporation, indicating the tolerance of tomato and pepper to ITCs released by the cover crops. Early-season yellow nutsedge control from Brassicaceae cover crops was ≤ 53% at 2 wk after transplanting and declined to ≤ 18% later in the season. This research demonstrates that Brassicaceae cover crops have marginal potential for early-season weed control and cannot be used as a weed control practice in commercial tomato and bell pepper production.