The comparison of cytotoxic and genotoxic activities of glucosinolates, isothiocyanates, and indoles.

Chemopreventive properties of Brassica vegetables are attributed mainly to their characteristic compounds-glucosinolates (GLs) and their main hydrolysis products-isothiocyanates (ITCs) and indoles. In this study, we compared antiproliferative activity (MTT test in HT29 cells) and genotoxic effects (comet assay in HT29 cells and restriction analysis in a cell-free system) of three GLs (sinigrin (SIN), glucotropaeolin (GTL), and glucobrassicin (GLB)) with that of their major degradation products. Intact GLs did not exhibit cytotoxic activity, possibly due to their limited bioavailability. However, in the presence of myrosinase (MYR), GLs gained the ability to inhibit HT29 cells' growth. The addition of MYR caused the hydrolysis of GLs to the corresponding ITCs or indoles, i.e. compounds that show stronger biological activity than parent GLs. Pure ITC/indole solutions showed the strongest antiproliferative activity. Based on the results of restriction analysis, it was found that GLs to a greater extent than ITCs caused DNA modification in a cell-free system. In the case of GLs, metabolic activation by the S9 fraction increased this effect, and at the same time changed the preferential binding site from the area of base pairs AT to GC base pairs. Of all compounds tested, only benzyl ITC caused DNA damage detectable in the comet assay, but it required relatively high concentrations.

Chemical Aspects of Biological Activity of Isothiocyanates and Indoles, the Products of Glucosinolate Decomposition.

There is growing evidence that cancer chemoprevention employing natural, bioactive compounds may halt or at least slow down the different stages of carcinogenesis. A particularly advantageous effect is attributed to derivatives of sulfur-organic phytochemicals, such as glucosinolates (GLs) synthesized mainly in Brassicaceae plant family. GLs are hydrolysed enzymatically to bioactive isothiocyanates (ITC) and indoles, which exhibit strong anti-inflammatory and anti-carcinogenic activity. Highly bioavailable electrophilic ITC are of particular interest, as they can react with nucleophilic groups of important biomolecules to form dithiocarbamates, thiocarbamates and thioureas. These modifications seem responsible for the chemopreventive activity, but also for genotoxicity and mutagenicity. It was documented that ITC can permanently bind to important biomolecules such as glutathione, cytoskeleton proteins, transcription factors NF-κB and Nrf2, thiol-disulfide oxidoreductases, proteasome proteins or heat shock proteins. Furthermore, ITC may also affect epigenetic regulation of gene expression, e.g. by inhibition of histone deacetylases. Some other derivatives of glucosinolates, especially indoles, are able to form covalent bonds with nucleobases in DNA, which may result in genotoxicity and mutagenicity. This article summarizes the current state of knowledge about glucosinolates and their degradation products in terms of possible interactions with reactive groups of cellular molecules.

The extended version of restriction analysis approach for the examination of the ability of low-molecular-weight compounds to modify DNA in a cell-free system.

One of the primary requirements in toxicology is the assessment of ability of chemicals to induce DNA covalent modification. There are several well-established methods used for this purpose such as (32)P-Postlabeling or HPLC-MS. However, all of these approaches have difficult to overcome limitations, which prevents their use in genotoxin screening. Here, we describe the simple protocol exploiting specificity of restriction enzymes for the detection of DNA modification. It uses a specifically designed DNA amplicon, which contains two restriction sites recognized by Tru1I or MspI/HapII endonucleases. Modification of a restriction site abolishes its recognition and thus cleavage by the corresponding enzyme. The inhibition of cleavage indicates the occurrence of DNA modification of the restriction site(s), simultaneously pointing at the kind of base pairs (AT or GC) involved in DNA adduct formation. Previously, the application of this method was demonstrated for two antitumor compounds. Current study shows the extended version, that includes different ways of activation of tested compounds. Moreover, we propose an array of applications being of interest in toxicological research such as monitoring the kinetics of DNA adduct formation, detection of oxidative DNA damage, as well as assessment of the ability of antioxidative phytochemicals to prevent the latter DNA lesions.

The influence of selenium addition during germination of Brassica seeds on health-promoting potential of sprouts.

The correlation among selenium uptake, the content of bioactive compounds in sprouts, and biological activities triggered in cultured human cells by sprout extracts was investigated. Seeds of Brassica crops and rye were treated with SeO2 water solution. The selenium levels in sprouts increased from 1.0-4.1 to 53.3-382 µg/g dw with no influence on plant physiology according to the indices used. Neither the composition of glucosinolates (GL) in Brassica sprouts nor the myrosinase activity nor the composition of GL breakdown lipophilic products were significantly affected. In all Brassica sprouts, conversion to health-promoting isothiocyanates (ITC) and indoles corresponded to only 1% of total GLs. Low ITC concentration may explain observed lack of induction of glutathione S-transferases (GST) and quinone oxidoreductase (NQO) detoxifying enzymes in HT29 cells exposed to sprout extracts. The insignificant impact on cell growth and genome function suggests that Brassica sprouts may be safe vehicle of selenium to combat its dietary deficiency.

Isothiocyanates may chemically detoxify mutagenic amines formed in heat processed meat.

Meat consumption represents a dietary risk factor increasing the incidence of common cancers, probably due to carcinogenic amines (HAAs) formed upon meat heating. Interestingly, cancers whose incidence is increased by meat consumption, are decreased in populations consuming brassica vegetables regularly. This inverse correlation is attributed to brassica anticarcinogenic components, especially isothiocyanates (ITCs) that stimulate detoxification of food carcinogens. However, ITC reactivity towards amines generating stable thioureas, may also decrease mutagenicity of processed meat. We confirmed here that combining meat with cabbage (fresh or lyophilized), in proportions found in culinary recipes, limited by 17-20% formation of HAAs and significantly lowered mutagenic activity of fried burgers. Moreover, MeIQx mutagenicity was lowered in the presence of ITCs, as well as for synthetic ITC-MeIQx conjugates. This suggests that formation of thioureas could lead to chemical detoxification of food carcinogens, reducing the cancer risk associated with meat consumption.