GLUCOSINOLATES AND THEIR BIOLOGICAL FUNCTION

IV. GLUCOSINOLATES AND THEIR BIOLOGICAL FUNCTION

A. Flavor Components Glucosinolates are sulfur-containing glycosides and are some of the most

abundant secondary metabolites in Brassica species; they function in plant insect interactions and as feeding deterrents for plant herbivores. These very same phytochemicals are also recognized by humans, particularly the ITCs, as the characteristic flavor components associated with cruciferous vegetables and salad crops. During tissue damage ITCs are often released and can be recognized by their often spicy and pungent aroma (42). In watercress, Ror- ripa nasturtium aquaticum , the primary metabolite is the volatile h-phenyl- ethyl isothiocyanate (PEITC), which gives this plant a characteristic hot

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pungent flavor. In contrast, broccoli, Brassica oleracea L. var. italica, the most widely consumed Brassica, produces the nonvolatile, N-methylsulphi- nylalkyl isothiocyanates giving broccoli its mild flavor. The bitter flavor associated with brussels sprouts, B. oleracea L. var. gemmifera, is proposed to

be due to hydrolysis of 2-hydroxy-3-butenyl and 2-propenyl glucosinolates; however, interaction with other phytochemical components may also con- tribute.

B. Antimicrobial Properties of Dietary Isothiocyanates The antibacterial effects of ITCs have been described on a number of

occasions. The inhibitory effects of arylalkyl ITCs on the bacteria Escherichia coli and Bacillus subtilis are attributed to their reaction with cellular thiol groups disrupting cellular homeostasis. Indeed, the inhibition of polypeptide synthesis in the cell-free system of E. coli was attributed to the inactivation of ribosomes (43). In a study by Ono et al. identification of the active antimi- crobial component in wasabi 6-methylsulfinylhexyl isothiocyanate was shown to have strong activity toward E. coli and Staphylococcus aureus (44). A comparison of the antibacterial properties of allyl isothiocyanate (AITC) with several antibacterial agents such as streptomycin, penicillin, and poly- mixin B has also been described. AITC induced a significant reduction in viability associated with the loss of membrane integrity in the bacteria S. montevideo , E. coli, and Listeria monocytogenes and was comparable to antibiotic treatments (45). More recent work conducted by Fahey and colleagues demonstrated the bacteriostatic effects of sulforaphane, a major constituent of broccoli, on the pathogenic bacterium Helicobacter pylori. The development of gastric and peptic ulcers induced by infection with H. pylori can often develop to more chronic disorders such as gastric cancer. Sulfo- raphane had a potent bacteriostatic effect against three reference strains and

45 clinical isolates of H. pylori. More promising was the fact that these properties were independent of H. pylori resistance to conventional anti- biotics (46). Similarly, antifungal activities have been observed for AITC, 5- methylthiopentyl, 3-methylsulfinylpropyl, and PEITC against Aspergillus niger , Penicillin cyclopium, and Rhizopus oryzae (47). However, whether these antifungal properties have any significance to human health has yet to be properly addressed.

C. Antinutritional Effects of Glucosinolate Hydrolytic Products It cannot be ignored that the beneficial effects of consuming cruciferous

vegetables is important in the proposed prevention of chronic human disease. It must also be noted that deleterious effects can also be attributed to high ex- posure to ITCs and other GSL hydrolytic products. In addition to acting as

372 Rose et al.

feeding deterrents for herbivores, some GSL metabolites also show goitro- genic activity in mammals. For example, the use of oilseed rape meal as animal feed was severally hampered owing to high levels of 2-hydroxy-3-butenyl glucosinolate. During GSL degradation ( ) 5-vinyloxazolidine-2-thione can

be formed, or in the presence of ESP 1-cyano-2-hydroxy-3-butene, both compounds can have a deleterious effects in mammals (Fig. 1b). Indeed, high exposure of livestock to plants containing high levels of both these com- pounds can result in the development of enlarged thyroids, stunted growth, and abnormalities of the liver and kidneys (21,48,49). These abnormalities are suggested to be a result of the impairment of thyroid function by inhibition of thyroxine synthesis. Fortunately, selective breeding has reduced the levels of hydroxyalkenyl GSLs in oilseed rape and has eliminated this problem. No evidence as yet has shown that cruciferous vegetables have a goitrogenic effect in humans. Volunteers consuming 150 g of brussels sprouts showed no impairment of thyroid function as assessed by thyroid hormone levels (50).

The toxicity of ITCs in mammals has also been the subject of several investigations, albeit at levels that are generally not attainable in the human diet. The promoting effects of AITC, benzyl isothiocyanate (BITC), PEITC, and their mercapturic acid metabolites in rat urinary bladder carcinogenicity have been described. Rats pretreated with the carcinogens diethylnitrosamine and N-butyl-N-(4-hydroxybutyl)nitrosamine showed a significant increase in the incidence of urinary bladder carcinomas after feeding with either PEITC or BITC in the postinitiation phase (51,52). BITC can also induce chromo- somal aberrations, sister chromatid exchange, and DNA strand breaks in cultured cells (53). The significance of these findings will be discussed later. Genotoxic effects of AITC, BITC, and PEITC have also been described using in vitro and in vivo techniques (54,55). Micronucleus induction assays in HepG2 cells and differential DNA repair assays in the bacterium E. coli indicate AITC to be strongly genotoxic albeit to a greater extent in bacterial systems. Similar genotoxic effects in bacteria have also been described for Brassica vegetable extracts. Eight different Brassica vegetables, including broccoli, cabbage, and brussels sprouts, were assessed on their ability to induce point mutations in the Salmonella strains TA98 and TA100, repairable DNA damage in E. coli, and clastogenic effects in mammalian cells. Induction of chromosomal aberrations and loss of cell viability were observed in mammalian cells and attributed to the GSL and ITC constituents (56). Likewise, a proposed mechanism for the genotoxic effects of AITC involving the generation of free radicals has been described. The generation of the oxidative DNA damage marker 8-oxo-7,8-dihydro-2V-deoxyguanosine in the presence of Cu 2+ by AITC in HL-60 cells is suggested as having a possible role in carcinogenesis. However, its role in cytotoxicity and tumor promotion has not been determined (57).

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Many of the toxic effects that have been described in the above studies may well be attributed to the ability of ITCs to induce apoptosis. Significant permutations in DNA damage and cellular function are often associated with the mediation of programmed cell death. Indeed, recent work has shown that DNA-damaging agents can initiate the apoptotic cascade and that this process may have a beneficial effect in removing neoplastic cells.