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.     

Mechanism of oxidative DNA damage induced by carcinogenic allyl isothiocyanate

Several isothiocyanates have been proposed as promising chemopreventive agents for human cancers. However, it has been reported that allyl isothiocyanate exhibit carcinogenic potential, and benzyl isothiocyanate and phenethyl isothiocyanate have tumor-promoting activities. We investigated whether these isothiocyanates could cause DNA damage, using (32)P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. Allyl isothiocyanate caused Cu(II)-mediated DNA damage and formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) more strongly than benzyl and phenethyl isothiocyanates. Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited Cu(II)-mediated DNA damage by these isothiocyanates, suggesting involvement of H(2)O(2) and Cu(I). Isothiocyanates induced DNA damage frequently at thymine and cytosine residues in the presence of Cu(II). A UV-visible spectroscopic study revealed an association between the generation of superoxide and the yield of SH group from isothiocyanates. Furthermore, the yield of 8-oxodG formation was correlated with their superoxide-generating ability. Allyl isothiocyanate significantly induced 8-oxodG formation in HL-60 cells, but not in H(2)O(2)-resistant HP100 cells, suggesting the involvement of H(2)O(2) in cellular DNA damage. We conclude that oxidative DNA damage may play important roles in carcinogenic processes induced by allyl isothiocyanate.     

Sulforaphane and its analogues inhibit CYP1A1 and CYP1A2 activity induced by benzo[a]pyrene

CYP1A1 and CYP1A2 enzymes metabolize polycyclic aromatic hydrocarbons (PAHs) to the reactive oxyderivatives. PAHs can induce the activity of both enzymes, which increases its conversion and enhances risk of carcinogenesis. Thus, the inhibition of CYP enzymes is recognized as a cancer chemoprevention strategy. A well‐known group of chemopreventive agents is isothiocyanates, which occur naturally in Brassica vegetables. In this paper, a naturally occurring sulforaphane and its two synthetic analogues isothiocyanate‐2‐oxohexyl and alyssin were investigated. The aim of the study was to determine whether the differences in the isothiocyanate structure change its ability to inhibit CYP1A1 and CYP1A2 activity induced by benzo[a]pyrene in HepG2 and Mcf7 cells. Also a mechanistic study was performed including isothiocyanates' influence on CYP1A1 and CYP1A2 catalytic activity, enzymatic protein level, and AhR translocation. It was shown that both enzymes were significantly induced by benzo[a]pyrene, and isothiocyanates were capable of decreasing the induced activity. The inhibitory properties depend on the types of isothiocyanate and enzyme. In general, CYP1A2 was altered in the more meaningful way than CYP1A1 by isothiocyanates. Sulforaphane exhibited weak inhibitory properties, whereas both analogues were capable of inhibiting BaP‐induced activity with the similar efficacy. The mechanistic study revealed that analogues decreased the CYP1A2 activity via the protein‐level reduction and CYP1A1 directly. The results indicate that isothiocyanates can be considered as potent chemopreventive substances and the change in the sulforaphane structure increases its chemopreventive potency. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:18–28, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20259     

Mitochondrial peroxiredoxin 3 is rapidly oxidized in cells treated with isothiocyanates

Isothiocyanates are phytochemicals with anti-cancer properties that include the ability to trigger apoptosis. A substantial body of evidence suggests that reaction of the electrophilic isothiocyanate moiety with cysteine residues in cellular proteins and glutathione accounts for their biological activity. In this study we investigated the effect of several different isothiocyanates on the redox states of the cysteine-dependent peroxiredoxins (Prx) in Jurkat T lymphoma cells, and compared this to known effects on the selenoprotein thioredoxin reductase, glutathione reductase and intracellular GSH levels. Interestingly, oxidation of mitochondrial Prx3 could be detected as early as 5 min after exposure of cells to phenethyl isothiocyanate, with complete oxidation occurring at doses that only had small inhibitory effects on total cellular thioredoxin reductase and glutathione reductase activities. Peroxiredoxin oxidation was specific to the mitochondrial isoform with cytoplasmic Prx1 and Prx2 maintained in their reduced forms at all analyzed time points and concentrations of isothiocyanate. Phenethyl isothiocyanate could react with purified Prx3 directly, but it did not oxidize Prx3 or promote its oxidation by hydrogen peroxide. A selection of aromatic and alkyl isothiocyanates were tested and while all lowered cellular GSH levels, only the isothiocyanates that caused Prx3 oxidation were able to trigger cell death. We propose that pro-apoptotic isothiocyanates selectively disrupt mitochondrial redox homeostasis, as indicated by Prx3 oxidation, and that this contributes to their pro-apoptotic activity.     

The role of Sulforaphane in cancer chemoprevention and health benefits: a mini-review

Cancer is a multi-stage process resulting from aberrant signaling pathways driving uncontrolled proliferation of transformed cells. The development and progression of cancer from a premalignant lesion towards a metastatic tumor requires accumulation of mutations in many regulatory genes of the cell. Different chemopreventative approaches have been sought to interfere with initiation and control malignant progression. Here we present research on dietary compounds with evidence of cancer prevention activity that highlights the potential beneficial effect of a diet rich in cruciferous vegetables. The Brassica family of cruciferous vegetables such as broccoli is a rich source of glucosinolates, which are metabolized to isothiocyanate compounds. Amongst a number of related variants of isothiocyanates, sulforaphane (SFN) has surfaced as a particularly potent chemopreventive agent based on its ability to target multiple mechanisms within the cell to control carcinogenesis. Anti-inflammatory, pro-apoptotic and modulation of histones are some of the more important and known mechanisms by which SFN exerts chemoprevention. The effect of SFN on cancer stem cells is another area of interest that has been explored in recent years and may contribute to its chemopreventive properties. In this paper, we briefly review structure, pharmacology and preclinical studies highlighting chemopreventive effects of SFN.     

Micronucleus formation and induction of apoptosis by different isothiocyanates and a mixture of isothiocyanates in human lymphocyte cultures

Isothiocyanates (ITCs) are the main sulfur-containing metabolites found in cruciferous vegetables. There is evidence that some ITCs may act as chemopreventive agents against different tumor types and induce apoptosis and modulate cell-cycle progression of highly proliferative cancer cells. However, there are also studies reporting genotoxic or co-carcinogenic effects for some ITCs, such as benzyl ITC and phenyl ITC. Since selectivity for transformed cells and absence of genotoxicity for healthy cells are important pre-requisites for new chemopreventive agents, we investigated micronucleus formation and induction of apoptosis by 4-(methylthio)butylisothiocyanate (MTBITC), sulforaphane and a mixture of ITCs in human T-lymphocyte cultures. We demonstrate that MTBITC, sulforaphane and the mixture of ITCs did not induce micronuclei. Moreover, sulforaphane induced a dose-dependent increase in the number of apoptotic cells, which was significant at the highest concentration tested (30 microM) (41% versus 18% in the untreated samples, P<0.05). The mixture of ITCs presented a trend similar to that found for sulforaphane. In fact, the mixture of ITCs was able to induce a dose-dependent increase in the percentage of apoptotic cells, which reached a maximum value at the concentration of 13 microg/ml (46% versus 19% in control samples, P<0.05). Induction of apoptosis was not observed in cultures treated with MTBITC. Our results suggest that different ITCs can have different effects. Moreover, although the mixture of glucosinolates (GLs) used in the present study does not reflect the exact composition of broccoli, our findings demonstrate that the quantitative effects of a single, specific ITC can be significantly different from those of an ITC mixture, where other ITCs of the mixture contribute to the outcome observed.     

Effect of naturally occurring organosulfur compounds on nitric oxide production in lipopolysaccharide-activated macrophages

Excessive nitric oxide (NO) production is involved in cellular injury and possibly in the multistage process of carcinogenesis. In this study, we investigated the effect of organosulfur compounds (S-allyl cysteine, allyl sulfide, diallyl disulfide, allyl isothiocyanate, phenyl isothiocyanate, and benzyl isothiocyanate) that are found in allium or cruciferous vegetables on NO production in J774.1 macrophages activated with lipopolysaccharide (LPS). Diallyl disulfide, allyl, phenyl, and benzyl isothiocyanates inhibited NO production, as evaluated by nitrite formation at 25 microM. Allyl and benzyl isothiocyanates, the most active of the six organosulfur compounds, exhibited dose-dependent inhibition and had IC(50) values of 1.6 and 2.7 microM, respectively. Western blot analysis suggested that suppression of the induction of inducible NO synthase (iNOS) expression is responsible for the inhibition of NO production by allyl and benzyl isothiocyanates. In contrast, these isothiocyanates increased LPS-stimulated tumor necrosis factor alpha (TNF-alpha) release, suggesting their selective action on genes activated by LPS. Our results demonstrate that certain organosulfur compounds inhibit NO synthesis in LPS-activated macrophages, and the inhibitory effect may be a significant component of their anticarcinogenic activity.     

Cell death induction by isothiocyanates and their underlying molecular mechanisms

An important and promising group of compounds that have a chemopreventive property are organosulfur compounds, such as isothiocyanates (ITCs). In recent years, it has been shown that ITCs induce apoptosis in various cancer cell lines and experimental rodents. During the course of apoptosis induction by ITC, multiple signal-transduction pathways and apoptosis intermediates are modulated. We have also clarified the molecular mechanism underlying the relationship between cell cycle arrest and apoptosis induced by benzyl isothiocyanate (BITC), a major ITC compound isolated from papaya. The exposure of cells to BITC resulted in the inhibition of the G2/M progression that coincided with not only the up-regulated expression of the G2/M cell cycle arrest-regulating genes but also the apoptosis induction. The experiment using the phase-specific synchronized cells demonstrated that the G2/M phase-arrested cells are more sensitive to undergoing apoptotic stimulation by BITC than the cells in other phases. We identified the phosphorylated Bcl-2 as a key molecule linking the p38 MAPK-dependent cell cycle arrest with the JNK activation by BITC. We also found that BITC induced the cytotoxic effect more preferentially in the proliferating normal human colon epithelial cells than in the quiescent cells. Conversely, treatment with an excessive concentration of BITC resulted in necrotic cell death without DNA ladder formation. This review addresses the biological impact of cell death induction by BITC as well as other ITCs and the involved signal transduction pathways.     

Transthiocarbamoylation of proteins by thiolated isothiocyanates

Isothiocyanates, membrane-permeable electrophiles that form adducts with thiols, have been suggested to have important medical benefits. Here we shed light on isothiocyanate-thiol conjugates and studied their electrophilic potential transferring an isothiocyanate moiety to cellular proteins. When we examined the effect of sulfhydryl molecules on cellular response induced by 6-methylsulfinylhexyl isothiocyanate (6-HITC), an analog of sulforaphane isolated from broccoli, we observed significant induction of heme oxygenase-1 by 6-HITC even in the presence of N-acetyl-L-cysteine or glutathione (GSH). In addition, the authentic 6-HITC-β-mercaptoethanol (6-HITC-ME) conjugate markedly up-regulated the enzyme expression, suggesting the electrophilic potential of thiolated isothiocyanates. To gain a chemical insight into the cellular response induced by thiolated isothiocyanates, we studied the occurrence of transthiocarbamoylation of sulfhydryl molecules by 6-HITC-ME and observed that, upon incubation of 6-HITC-ME with GSH, a single product corresponding to the GSH conjugate of 6-HITC was generated. To test the functional ability of thiolated isothiocyanates to thiocarbamoylate proteins in living cells, we designed a novel probe, combining an isothiocyanate-reactive group and an alkyne functionality, and revealed that the transthiocarbamoylation of proteins occurred in the cells upon exposure to 6-HITC-ME. The target of thiocarbamoylation included heat shock protein 90 β (Hsp90β), a chaperone ATPase of the Hsp90 family implicated in protein maturation and targeting. To identify the sites of the Hsp90β modification, we utilized nano-LC/MALDI-TOF MS/MS and suggested that a thiol group on the peptide containing Cys-521 reacted with 6-HITC, resulting in a covalent adduct in a 6-HITC-treated recombinant Hsp90β in vitro. The site-selective binding to Cys-521 was supported by in silico modeling. Further study on the thiocarbamoylation of Hsp90β suggested that the formation of 6-HITC-Hsp90β conjugate might cause activation of heat shock factor-1, rapidly signaling a potential heat shock response. These data suggest that thiolated isothiocyanates are an active metabolite that could contribute to cellular responses through transthiocarbamoylation of cellular proteins.     

Mortality and behavior in Heterodera glycines juveniles following exposure to isothiocyanate compounds

For this report, we examined the toxic effects of three plant-derived isothiocyanate compounds on second-stage juveniles (J2) of Heterodera glycines. We found significant differences among compounds in the concentration required to affect nematodes, according to mortality and behavioral measurements. The concentrations required to affect behavior were significantly lower than those required for mortality. Both mortality and behavioral measurements were used to investigate whether nematodes in a quiescent state display decreased sensitivity to isothiocyanates compared with actively moving nematodes. Mortality measurements revealed that quiescent nematodes were significantly less sensitive to isothiocyanates than active nematodes. All behavioral measurements following exposure to benzyl- and phenyl isothiocyanate showed significant differences in sensitivity between quiescent and active nematodes. However, significant differences between quiescent and active nematodes were observed in only one of the five behavioral measurements following exposure to allyl isothiocyanate. These results expand the list of plant-derived compounds toxic to H. glycines and illustrate the impact of behavioral quiescence on nematode sensitivity to exogenous toxins.     

Isothiocyanates inhibit fungal pathogens of potato in in vitro assays

Aims

This study aimed to examine the effects of seven different isothiocyanates against the growth and development of three important soil borne potato pathogens, (Colletotrichum coccodes, Rhizoctonia solani and Helminthosporium solani).

Methods

The study was carried out using an agar diffusion assay. The radial growth of fungal pathogens grown on agar containing different ITCs at a range of concentrations was compared to that of growth on control agar plates that did not contain ITCs.

Results

Results varied depending on the specific isothiocyanate incorporated into the agar. They ranged from those which showed a significant effect on fungal growth to those which appeared to have little or no effect. Where a suppressive effect was observed, due to the presence of the isothiocyanate, fungal colony growth decreased as the concentration of the incorporated isothiocyanate increased.

Conclusions

Results from this study indicate that fungal growth can be inhibited by exposure to ITCs. However the results observed are specific to the ITC structure and exposure concentration.     

The responses of the cabbage seed weevil Ceutorhynchus assimilis to volatile compounds from oilseed rape in a linear track olfactometer

The cabbage seed weevil, Ceutorhynchus assimilis Payk. [syn. Ceutorhynchus obstrictus (Marsham)] (Coleoptera: Curculionidae), a crucifer-feeding insect, is a pest of oilseed rape (Brassica napus L.). It is known to be attracted by isothiocyanates, crucifer-specific volatiles that are metabolites of the glucosinolates. The responses of this insect to other electrophysiologically-active volatiles from rape were tested in a linear track olfactometer. Attraction was demonstrated to nitriles (phenylacetonitrile, 4-pentenenitrile and 5-hexenenitrile), which are also glucosinolate metabolites, and to volatiles emitted by a wider spectrum of plant families ((Z)-3-hexen-1-ol and methyl salicylate). Combination of an isothiocyanate mixture with phenylacetonitrile increased attraction, but there was no such increase when the isothiocyanate mixture was combined with methyl salicylate. A mixture of 23 volatiles, emulating an attractive air-entrainment extract of oilseed rape, was not significantly attractive, although a high proportion of weevils (60%) turned towards it. The potential of these volatiles for inclusion into an isothiocyanate-based monitoring system is discussed.     

Analysis of Volatile Components in Essential Oil of Upland Wasabi and Their Inhibitory Effects on Platelet Aggregation

Inhibition of platelet aggregation was found in the essential oils extracted from the various parts of upland wasabi (Wasabia japonica forma terrestris Hisauti). The antiplatelet activity increased in the order of leaves, rhizomes, petioles, and roots. To examine the inhibitory principles in upland wasabi oil, components in each part of the essential oil were identified and measured. Most of the volatile components in upland wasabi oils were ω-alkenyl isothiocyanates and ω-methylthioalkyl isothiocyanates, allyl isothiocyanate being the main component in every oil. Essential oils from roots and petioles contained rather large amounts of ω-methylthioalkyl isothiocyanates. Although every authentic isothiocyanate inhibited platelet aggregation, ω-methylthioalkyl isothiocyanates were more inhibitory, which explained the stronger inhibitory effects of essential oils from roots and petioles on platelet aggregation.     

Effect of Synthetic Hydroxy Isothiocyanates on a Bacterial Virus and DNA

The Effect of hydroxy isothiocyanates on a bacterial virus and M13 DNA was examined. Hydroxy-substituted phenyl and phenyl alkyl isothiocyanates, especially 2-(3,4-dihydroxyphenyl)ethyl isothiocyanate(IT-Dop) synthesized from dopamine, showed antiviral activity on φK. In transfection experiments with M13 mp DNA species, IT-Dop inhibited the single-stranded (SS) molecule more effectively than the double stranded replicative form (RF) DNA. These effects were dependent on reaction time, and on IT-Dop concentration. An additional experiment indicated that treatment with IT-Dop suppressed annealing (reassociation) of denatured DNA. These results indicate that IT-Dop reacts mildly with virus and SS DNA.     

Leaching of isothiocyanates through intact soil following simulated biofumigation

Biofumigation can be used as an alternative to conventional soil fumigation to control soil-borne pests. With biofumigation, plant tissue with a natural content of glucosinolates (cruciferous plants) is damaged and incorporated into the topsoil. When the plant tissue is damaged, the glucosinolates come into contact with the endogenous enzyme myrosinase, which catalyse the hydrolysis of glucosinolates into various products depending on the reaction conditions. Isothiocyanates are among the potential products formed from these reactions. We investigated if the isothiocyanates from rape plant material were leached through the soil to drain depth when a heavy rainstorm followed the biofumigation. We applied isothiocyanates from rape plant material (1,480 μmol m⁻²) to four large (0.6 m diameter, 1.0 m long) intact soil monoliths from a loamy and a sandy soil and conducted a leaching experiment under semi-field conditions. The soil monoliths were irrigated with 70–90 mm (10 mm h⁻¹) and the concentrations of three isothiocyanates (3-butenyl, 4-pentenyl and 2-phenethyl) were monitored in the leachate. Between 0 and 14.8 mmol isothiocyanates were leached for each mol of isothiocyanates applied during application of 70–90 mm irrigation. The distribution coefficient estimated from leached concentrations was 0.04–1.19 for 3-butenyl, 0.04–1.15 for 4-pentenyl isothiocyanate and 0.037–0.97 for 2-phenethyl isothiocyanate. The concentration of total isothiocyanates in the leachate was in the same order of magnitude as the LD₅₀ of isothiocyanates for sensitive aquatic organisms.     

Effect of synthetic hydroxy isothiocyanates on a bacterial virus and DNA

The Effect of hydroxy isothiocyanates on a bacterial virus and M13 DNA was examined. Hydroxy-substituted phenyl and phenyl alkyl isothiocyanates, especially 2-(3,4-dihydroxyphenyl)ethyl isothiocyanate(IT-Dop) synthesized from dopamine, showed antiviral activity on psiK. In transfection experiments with M13 mp DNA species, IT-Dop inhibited the single-stranded (SS) molecule more effectively than the double stranded replicative form (RF) DNA. These effects were dependent on reaction time, and on IT-Dop concentration. An additional experiment indicated that treatment with IT-Dop suppressed annealing (reassociation) of denatured DNA. These results indicate that IT-Dop reacts mildly with virus and SS DNA.     

Phytochemicals as anticancer and chemopreventive topoisomerase II poisons

Phytochemicals are a rich source of anticancer drugs and chemopreventive agents. Several of these chemicals appear to exert at least some of their effects through interactions with topoisomerase II, an essential enzyme that regulates DNA supercoiling and removes knots and tangles from the genome. Topoisomerase II-active phytochemicals function by stabilizing covalent protein-cleaved DNA complexes that are intermediates in the catalytic cycle of the enzyme. As a result, these compounds convert topoisomerase II to a cellular toxin that fragments the genome. Because of their mode of action, they are referred to as topoisomerase II poisons as opposed to catalytic inhibitors. The first sections of this article discuss DNA topology, the catalytic cycle of topoisomerase II, and the two mechanisms (interfacial vs. covalent) by which different classes of topoisomerase II poisons alter enzyme activity. Subsequent sections discuss the effects of several phytochemicals on the type II enzyme, including demethyl-epipodophyllotoxins (semisynthetic anticancer drugs) as well as flavones, flavonols, isoflavones, catechins, isothiocyanates, and curcumin (dietary chemopreventive agents). Finally, the leukemogenic potential of topoisomerase II-targeted phytochemicals is described.     

Glucosinolates and isothiocyanates in health and disease

Glucosinolates and isothiocyanates have both been objects of research for more than half a century. Interest in these unique phytochemicals escalated following the discovery that sulforaphane, an isothiocyanate from broccoli, potently induces mammalian cytoprotective proteins through the Keap1-Nrf2-ARE pathway. In parallel with the advances in understanding the molecular regulation of this pathway and its critical role in protection against electrophiles and oxidants, there have been increased efforts toward translating this knowledge to improve human health and combat disease. This review focuses on the animal studies demonstrating the beneficial effects of glucosinolates and isothiocyanates in models of carcinogenesis, and cardiovascular and neurological diseases, as well as on the intervention studies of their safety, pharmacokinetics, and efficacy in humans.