The synthesis of [phenyl‐2H5]gluconasturtiin and its metabolites for metabolic studies

The synthesis of a deuterium labelled glucosinolate, [²H₅]gluconasturtiin, is described starting from [²H₅]bromobenzene. The potential metabolites of the glucosinolate, namely the [phenyl‐²H₅]phenethyl isothiocyanate, nitrile, thiocyanate, amine and the mercapturic acid conjugate of phenethyl isothiocyanate are also described. This series of compounds has been prepared for use in feeding studies to examine the mammalian metabolism of gluconasturtiin and search for new biomarkers of exposure. Copyright © 2005 John Wiley & Sons, Ltd.     

硫代葡萄糖苷及异硫氰酸酯的抗癌和抗氧化作用进展

目的为硫代葡萄糖苷及异硫氰酸酯的合理利用、功能性保健产品开发和抗癌辅助药物的研制提供依据。方法归纳总结近10年硫代葡萄糖苷和异硫氰酸酯的文献,对其在植物体的分布、理化性质、抗癌和抗氧化机制等方面进行详细阐述。结果硫代葡萄糖苷及其水解产物异硫氰酸酯具有良好的抗癌和抗氧化活性,可以预防和治疗多种癌症,对于由空气污染引发的呼吸系统及肺部疾病也具有良好的疗效;硫代葡萄糖苷及异硫氰酸酯可以清除血管和机体的自由基,保护心脑血管,延缓衰老。结论硫代葡萄糖苷及异硫氰酸酯开发成功能性保健产品和抗癌辅助药具有广阔的市场前景。     

Genotoxicity studies of organically grown broccoli (Brassica oleracea var. italica) and its interactions with urethane,methyl methanesulfonate and 4-nitroquinoline-1-oxide genotoxicity in the wing spot test of Drosophila melanogaster

Broccoli (Brassica oleracea var. italica) has been defined as a cancer preventive food. Nevertheless, broccoli contains potentially genotoxic compounds as well. We performed the wing spot test of Drosophila melanogaster in treatments with organically grown broccoli (OGB) and co-treatments with the promutagen urethane (URE), the direct alkylating agent methyl methanesulfonate (MMS) and the carcinogen 4-nitroquinoline-1-oxide (4-NQO) in the standard (ST) and high bioactivation (HB) crosses with inducible and high levels of cytochrome P450s (CYPs), respectively. Larvae of both crosses were chronically fed with OGB or fresh market broccoli (FMB) as a non-organically grown control, added with solvents or mutagens solutions. In both crosses, the OGB added with Tween–ethanol yielded the expected reduction in the genotoxicity spontaneous rate. OGB co-treatments did not affect the URE effect, MMS showed synergy and 4-NQO damage was modulated in both crosses. In contrast, FMB controls produced damage increase; co-treatments modulated URE genotoxicity, diminished MMS damage, and did not change the 4-NQO damage. The high dietary consumption of both types of broccoli and its protective effects in D. melanogaster are discussed.     

A principal mechanism for the cancer chemopreventive activity of phenethyl isothiocyanate is modulation of carcinogen metabolism

Isothiocyanates are small molecules characterized by high chemical reactivity that allows them to interact readily with cellular constituents eliciting a plethora of biological activities. They are present exclusively in cruciferous vegetables, as glucosinolates, the intake of which has been associated with cancer chemoprevention. When the physical structure of these vegetables is disturbed, e.g. during mastication, the enzyme myrosinase is released and converts the glucosinolates to isothiocyanates (R–N=C=S), where R can be aliphatic or aromatic. Although sulforaphane, an aliphatic isothiocyanate, has received most attention worldwide, the most extensively studied aromatic isothiocyanate is phenethyl isothiocyanate (PEITC), and there are substantial differences in biological activity between the two sub-classes. In animal cancer models, PEITC effectively antagonized the carcinogenicity of chemicals, especially nitrosocompounds. A principal mechanism of their action is to protect the integrity of DNA by decreasing the levels of the genotoxic metabolites of chemical carcinogens. Extensive studies established that PEITC modulates the metabolism of the tobacco-specific carcinogenic nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by inhibiting its cytochrome P450-mediated bioactivation. Moreover, PEITC is a potent inducer of detoxification enzymes such as quinone reductase, glutathione S-transferase and glucuronosyl transferase. PEITC is rapidly absorbed and is characterized by a large bioavailability; C_max concentrations achieved in plasma after dietary intake are sufficient to modulate carcinogen metabolism. PEITC is primarily metabolized by glutathione conjugation and is excreted in the urine and bile as the mercapturate. The ability of PEITC to perturb carcinogen metabolism through modulation of cytochrome P450 and phase II detoxification enzymes is comprehensively and critically reviewed.     

Cruciferous Vegetables: Dietary Phytochemicals for Cancer Prevention

Relationships between diet and health have attracted attention for centuries; but links between diet andcancer have been a focus only in recent decades. The consumption of diet-containing carcinogens, includingpolycyclic aromatic hydrocarbons and heterocyclic amines is most closely correlated with increasing cancer risk.Epidemiological evidence strongly suggests that consumption of dietary phytochemicals found in vegetables andfruit can decrease cancer incidence. Among the various vegetables, broccoli and other cruciferous species appearmost closely associated with reduced cancer risk in organs such as the colorectum, lung, prostate and breast.The protecting effects against cancer risk have been attributed, at least partly, due to their comparatively highamounts of glucosinolates, which differentiate them from other vegetables. Glucosinolates, a class of sulphurcontainingglycosides, present at substantial amounts in cruciferous vegetables, and their breakdown productssuch as the isothiocyanates, are believed to be responsible for their health benefits. However, the underlyingmechanisms responsible for the chemopreventive effect of these compounds are likely to be manifold, possiblyconcerning very complex interactions, and thus difficult to fully understand. Therefore, this article providesa brief overview about the mechanism of such compounds involved in modulation of carcinogen metabolisingenzyme systems.     

Coselected genes determine adaptive variation in herbivore resistance throughout the native range of Arabidopsis thaliana

The “mustard oil bomb” is a major defense mechanism in the Brassicaceae, which includes crops such as canola and the model plant Arabidopsis thaliana. These plants produce and store blends of amino acid-derived secondary metabolites called glucosinolates. Upon tissue rupture by natural enemies, the myrosinase enzyme hydrolyses glucosinolates, releasing defense molecules. Brassicaceae display extensive variation in the mixture of glucosinolates that they produce. To investigate the genetics underlying natural variation in glucosinolate profiles, we conducted a large genome-wide association study of 22 methionine-derived glucosinolates using A. thaliana accessions from across Europe. We found that 36% of among accession variation in overall glucosinolate profile was explained by genetic differentiation at only three known loci from the glucosinolate pathway. Glucosinolate-related SNPs were up to 490-fold enriched in the extreme tail of the genome-wide F_ST scan, indicating strong selection on loci controlling this pathway. Glucosinolate profiles displayed a striking longitudinal gradient with alkenyl and hydroxyalkenyl glucosinolates enriched in the West. We detected a significant contribution of glucosinolate loci toward general herbivore resistance and lifetime fitness in common garden experiments conducted in France, where accessions are enriched in hydroxyalkenyls. In addition to demonstrating the adaptive value of glucosinolate profile variation, we also detected long-distance linkage disequilibrium at two underlying loci, GS-OH and GS-ELONG. Locally cooccurring alleles at these loci display epistatic effects on herbivore resistance and fitness in ecologically realistic conditions. Together, our results suggest that natural selection has favored a locally adaptive configuration of physically unlinked loci in Western Europe.Both wild and cultivated plants face a great variety of natural enemies, including herbivores and pathogens, that can negatively impact their growth and yield (1, 2). To afford protection, plants use a wide array of defenses, such as the biosynthesis of toxic secondary metabolites. One such chemical defense is the glucosinolate-myrosinase system that is pervasive in the mustard family Brassicaceae, including crops such as canola and the model plant Arabidopsis thaliana (2–4). Glucosinolates (GSLs) form a diverse class of amino acid-derived thioglycosides. Myrosinases, a corresponding family of glycoside hydrolases, are sequestered from the GSLs in healthy plant tissue. Upon tissue rupture, myrosinases hydrolyze the GSLs to various products including isothiocyanates and nitriles (2), the specific structure and bioactivity of which is dependent upon the GSL side chain and the prevailing chemical conditions during hydrolysis (2). Although GSL hydrolysis products generally inhibit herbivory and the growth of pathogens, many pests have specialized adaptations to avoid toxic effects and use GSLs or their hydrolysis products as feeding or oviposition stimulants (5–7). Biosynthesis of methionine-derived GSLs in the model plant A. thaliana is characterized by three successive multistep phases of side-chain elongation, GSL core construction, and side-chain modification, involving dozens of genes (1, 7). Species in the Brassicaceae display extensive variation for GSL profiles (2, 5, 8–10). Intraspecific variation has been particularly well documented in accessions of A. thaliana, which produces blends of more than 30 GSL molecules (2, 11–13).A recent study showed that the GS-Elong locus, involved in side-chain elongation of GSLs, is correlated with the abundance of aphids along a longitudinal cline across Europe (14). The role of selection in generating this pattern has been supported with an experimental selection study under controlled conditions (14), although the selective role of particular natural enemies, including aphids, on A. thaliana in Europe is unknown. In addition, although the European cline in GS-Elong suggests that A. thaliana has been selected by herbivores to alter the length of its GSLs, no study has yet examined how well Arabidopsis chemotypes are defended in the field in Europe. Furthermore, little is known about how selection acts on other aspects of glucosinolate diversity. Here, we take a genomic perspective and explore how selection has shaped the loci regulating the biosynthesis and natural variation in a complex suite of GSLs across Europe.We characterized the relative concentrations of 22 methionine-derived GSLs in the leaves of 595 A. thaliana accessions collected from across its geographic range (15) (Table S1 and Dataset S1). Under controlled greenhouse conditions, natural accessions displayed genetic variation for the blend of molecules they produce. The plant genotype (accession) explained a significant proportion of the phenotypic variation for 16 out of the 22 molecules studied, with broad-sense heritabilities ranging from 5% to 73% (Fig. S1). The first principal component describing this variation explained 58% of the variation among accessions. This component was characterized by high positive loading scores for butenyls, pentenyls, and their respective hydroxylated products. The second principal component explained approximatively 25% of the variation and was mostly positively loaded with short-chain alkenyls, 2-propenyl (sinigrin) and 3-butenyl. Both components (hereafter GSL profile) displayed strong geographical clines (Fig. 1 and Table S2), with accessions from Western Europe containing far more alkenyl and hydroxyalkenyl GSLs than populations across Central Europe, Eastern Europe, and Sweden (Fig. 1 and Fig. S2).Open in a separate windowFig. 1.Map of methionine-derived GSL variation in Europe. The x and y axes correspond to longitude and latitude, respectively. Dots indicate collection sites, and the color reflects the score of each accession along the first (top map) and second (bottom map) principal component describing GSL profile variation. The gradient from blue to yellow represents an increase in alkenyl and hydroxyalkenyl GSLs (Fig. S2).We sought to identify regions of the genome important in shaping GSL profiles by completing a large genome-wide association (GWA) study with our 595 accessions. Each of these accessions has been genotyped for 197,763 single-nucleotide polymorphisms (SNPs) (15, 16). We estimated associations in a mixed-model framework that controlled for confounding due to population structure by including a matrix of kinship among accessions as a random effect (17, 18). Significance was determined by permuting phenotypes 10,000 times for each GSL molecule. A total of 474 significant associations were detected for 12 of the 22 individual GSL molecules used for mapping (Dataset S1). These significant associations represent 227 unique SNPs, all detected for multiple molecules (from 3 to 11). The three main regions corresponded to regions near confirmed GSL biosynthesis genes (Fig. 2 A and B, and Dataset S1), with our strongest association located on chromosome 4 near the GS-AOP/GS-OHP locus, composed of the genes AOP2 (AT4G03060, alkenyl hydroxalkyl producing 2) and AOP3 (AT4G03050) (hereafter “AOP region”) (11). Our second strongest association, located on chromosome 5, was situated near the GS-ELONG locus, containing the genes MAM1 (AT5G23010, methylthioalkylmalate synthase 1) and MAM3 (AT5G23020, methylthioalkylmalate synthase-like) (hereafter “MAM region”). Next, a region of chromosome 2 contained four significantly associated SNPs located within 6 kbp (kb) of the GS-OH locus (AT2G25450), which is known to regulate hydroxylation of alkenyl GSLs (hereafter the “GS-OH” region) (19). Associations near GS-OH were detected when mapping the leaf concentrations of progoitrin (2-hydroxy-3-butenyl), consistent with previous studies (1, 19), as well as napoleiferin (2-hydroxy-4-pentenyl). Although other SNPs distributed throughout the genome were significantly associated with GSL variation, these were primarily individual markers with no obvious candidates located within 20 kbp. The extensive genetic variation captured in our large sample of accessions allowed the identification of the GS-OH locus, which went undetected in a previous GWA study that included fewer accessions, but spanned a similar geographical range (20).Open in a separate windowFig. 2.Confirmed glucosinolate genes, GWA mapping, and F_ST scan. (A) Positions of confirmed genes from the methionine-derived GSL biosynthesis pathway along the five chromosomes of A. thaliana. (B) Manhattan plot of mapping results using EMMAX pooled from all 22 molecules analyzed. SNPs with significant association scores are marked in gold. (C) Wright’s fixation index (F_ST) scan along the Arabidopsis genome, based on 1,080 European accessions, divided into nine populations (15). The blue dotted line represents the 99.5% quantile of the F_ST distribution. SNPs significantly associated with GSL variation are marked in gold.Despite the previously unidentified detection of GS-OH, it was surprising that no other known GSL genes were identified. To explore the possibility that GSL loci were missed due to genetic/allelic heterogeneity or skewed allele frequencies, we performed genome-wide scans in regional mapping populations from France, Sweden, and the United Kingdom (15, 21). These scans identified two additional peaks in Sweden, but no genes related to GSL biosynthesis were located within 20 kbp. We also calculated both GSL ratios for compounds on adjacent steps of the biosynthetic pathway and composite GSLs (1, 22). This strategy also failed to identify GSL loci other than GS-ELONG, GS-AOP/GS-OHP, and GS-OH (see Dataset S1 for all association mapping results). Previous studies found variation among accessions for the conversion of methylthioalkyl to methylsulfinylalkyl and mapped several GS-OX loci using crosses (11, 23–25). We did not find significant associations near known flavin monooxygenases (FMO) genes, now known to constitute the GS-OX loci (25). Genetic heterogeneity at the FMO genes, known to have redundant functions, may explain our failure to detect them (21, 25). Overall, and in agreement with previous studies, our results suggest that only three loci explain most of the natural variation in GSL profiles, even though many genes have been implicated in the biosynthesis of aliphatic GSLs.The key loci governing variation in GSL profiles are located on three different chromosomes, raising the possibility that a genome-wide signature of selection could be detected. We investigated the degree of overlap between our 227 GSL-related SNPs and three published scans of selection using the RegMap panel (15) from which our accessions derived. Although neither the composite likelihood ratio of the allele frequency spectrum (26) nor the pairwise haplotype sharing test (27) showed significant overlap with GSL-associated SNPs, the 0.5% tail of the F_ST (28, 29) distribution displayed 28-fold enrichment for SNPs associated with GSL variation (Fig. 2C) and the 0.1% tail of the F_ST distribution displayed 490-fold enrichment. The MAM, GS-OH, and AOP regions all displayed high F_ST values, suggesting strong adaptive differentiation among populations. The fact that the genetics of this quantitative trait leaves a clear signature of selection across the genome suggests that variation in the GSL profile is an important adaptive trait.To confirm the adaptive role of the GSL profile, we conducted field experiments in Lille, France (i.e., in the west of Europe), a region enriched in the hydroxy-alkenyl GSLs that contribute most to natural variation in GSL profiles. First, we scored herbivore damage inflicted on the rosettes of 256 natural accessions in three common garden experiments over 2 successive years. Most of the rosette damage resulted from feeding by insect herbivores, although it is possible that molluscan herbivores also contributed despite efforts to exclude them (Fig. S3). Second, at the end of the field experiments, we estimated the total length of mature fruits produced by each plant, a proxy for lifetime fitness in this mostly selfing, annual species (30). This fitness estimate was highly heritable, with a broad-sense heritability of 51.28% (95% confidence interval, 44.76–58.22) across experiments (Tables S3 and S4).We explored the adaptive value of the GSL profile by investigating its relationship with herbivore damage and lifetime fitness across the experiments. The level of herbivore damage was negatively correlated with the principal component capturing most of the GSL profile variation among accessions (Spearman ρ = ?0.17; P = 0.008), despite GSLs being characterized on undamaged plants under greenhouse conditions (31). This indicates that accessions enriched in butenyls, pentenyls, and their hydroxylated derivatives were protected from herbivores in France. In a field study in Ohio, European accessions enriched in alkenyl GSLs were less protected (32). This disparity suggests herbivore communities vary in their GSL preference. The reduced herbivory that we detected translated to significantly higher lifetime fitness, even after including a covariate to control for “home vs. away” effects captured by the distance between Lille and the original collection site of the accessions (Table S5). The geographical distance to the collection site also had a significant, negative impact on plant fitness, consistent with previous studies showing adaptation to climate in natural populations of A. thaliana (33, 34).In addition to testing the influence of the GSL profile on fitness, we tested the effect of GS-OH, MAM, and AOP directly. To capture as much of the complex allelic variation present as possible (35, 36), we characterized the genetic variation at each locus with a pairwise genetic distance matrix (1 ? kinship, Fig. S4). We tested the effects of the three genetic distance matrices, as well as their two- and three-way interactions, on a matrix of fitness differences between accessions. Our regression also included a genetic distance matrix calculated for all other SNPs in the genome to guard against confounding due to population structure and/or adaptive genetic variation at other loci. The resulting model succeeded in explaining fitness differences among accessions (r² = 0.0969; P ≤ 0.00001). In particular, we detected a significant effect of the interaction between allelic differentiation at MAM and GS-OH on fitness, as well as a significant effect of the three-way interaction describing allelic differentiation at MAM, GS-OH, and AOP (37).

Table 1.

Multiple regression on matrices of fitness variation explained by the three GSL loci and their interactions

芥子中芥子碱类和硫代葡萄糖苷类成分化学稳定性和质量评价研究进展

芥子中主要化学成分为硫代葡萄糖苷类和芥子碱类。芥子碱类成分具有抗氧化、抗雄性激素、缓慢降压等多种药理活性;硫代葡萄糖苷类成分及其降解产物具镇咳平喘、抗氧化、抗癌、抗菌等功效。综述了芥子中硫代葡萄糖苷类和芥子碱类成分的化学组成、稳定性和质量控制方法的最新研究进展,以期对芥子药材及其相关产品的质量控制和合理开发利用提供科学指导。     

Glutathione and tryptophan metabolism are required for Arabidopsis immunity during the hypersensitive response to hemibiotrophs

The hypersensitive response (HR) is a type of strong immune response found in plants that is accompanied by localized cell death. However, it is unclear how HR can block a broad range of pathogens with different infective modes. In this study, we report that γ-glutamylcysteine synthetase GSH1, which is critical for glutathione biosynthesis, and tryptophan (Trp) metabolism contribute to HR and block development of fungal pathogens with hemibiotrophic infective modes. We found that GSH1 is involved in the penetration2 (PEN2)-based entry control of the nonadapted hemibiotroph Colletotrichum gloeosporioides. However, Arabidopsis mutants specifically defective in entry control terminated further growth of the pathogen in the presence of HR cell death, whereas gsh1 mutants supported pathogen invasive growth in planta, demonstrating the requirement of GSH1 for postinvasive nonhost resistance. Remarkably, on the basis of the phenotypic and metabolic analysis of Arabidopsis mutants defective in Trp metabolism, we showed that biosynthesis of Trp-derived phytochemicals is also essential for resistance to C. gloeosporioides during postinvasive HR. By contrast, GSH1 and these metabolites are likely to be dispensable for the induction of cell death during postinvasive HR. Furthermore, the resistance to Ralstonia solanacearum 1/resistance to Pseudomonas syringae 4 dual Resistance gene-dependent immunity of Arabidopsis to the adapted hemibiotroph shared GSH1 and cytochromes P450 CYP79B2/CYP79B3 with postinvasive nonhost resistance, whereas resistance to P. syringae pv. maculicola 1 and resistance to P. syringae 2-based Resistance gene resistance against bacterial pathogens did not. These data suggest that the synthesis of glutathione and Trp-derived metabolites during HR play crucial roles in terminating the invasive growth of both nonadapted and adapted hemibiotrophs.     

Phytochemicals in Cancer Prevention and Therapy: Truth or Dare?

A voluminous literature suggests that an increase in consumption of fruit and vegetables is a relatively easy and practical strategy to reduce significantly the incidence of cancer. The beneficial effect is mostly associated with the presence of phytochemicals in the diet. This review focuses on a group of them, namely isothiocyanate, curcumin, genistein, epigallocatechin gallate, lycopene and resveratrol, largely studied as chemopreventive agents and with potential clinical applications. Cellular and animal studies suggest that these molecules induce apoptosis and arrest cell growth by pleiotropic mechanisms. The anticancer efficacy of these compounds may result from their use in monotherapy or in association with chemotherapeutic drugs. This latter approach may represent a new pharmacological strategy against several types of cancers. However, despite the promising results from experimental studies, only a limited number of clinical trials are ongoing to assess the therapeutic efficacy of these molecules. Nevertheless, the preliminary results are promising and raise solid foundations for future investigations.     

Dealing with variability in food production chains: a tool to enhance the sensitivity of epidemiological studies on phytochemicals

Summary. Background: Many epidemiological studies have tried to associate the intake of certain food products with a reduced risk for certain diseases. Results of these studies are often ambiguous, conflicting, or show very large deviations of trends. Nevertheless, a clear and often reproduced inverse association is observed between total vegetable and fruit consumption and cancer risk. Examples of components that have been indicated to have a potential protective effect in food and vegetables include antioxidants, allium compounds and glucosinolates. Aim: The food production chain can give a considerable variation in the level of bioactive components in the products that are consumed. In this paper the effects of this variability in levels of phytochemicals in food products on the sensitivity of epidemiological studies are assessed. Methods: Information on the effect of variation in different steps of the food production chain of Brassica vegetables on their glucosinolate content is used to estimate the distributions in the levels in the final product that is consumed. Monte Carlo simulations of an epidemiological cohort study with 30,000 people have been used to assess the likelihood of finding significant associations between food product intake and reduced cancer risk. Results: By using the Monte Carlo simulation approach, it was shown that if information on the way of preparation of the products by the consumer was quantified, the statistical power of the study could at least be doubled. The statistical power could be increased by at least a factor of five if all variation of the food production chain could be accounted for. Conclusions: Variability in the level of protective components arising from the complete food production chain can be a major disturbing factor in the identification of associations between food intake and reduced risk for cancer. Monte Carlo simulation of the effect of the food production chain on epidemiological cohort studies has identified possible improvements in the set up of such studies. The actual effectiveness of food compounds already identified as cancer-protective by current imprecise methods is likely to be much greater than estimated at present. Received: 15 July 2002, Accepted: 26 January 2003 Correspondence to: Matthijs Dekker