Glucoraphanin, the bioprecursor of the widely extolled chemopreventive agent sulforaphane found in broccoli, induces phase-I xenobiotic metabolizing enzymes and increases free radical generation in rat liver.

Epidemiological and animal studies linking high fruit and vegetable consumption to lower cancer risk have strengthened the belief that long-term administration of isolated naturally occurring dietary constituents could reduce the risk of cancer. In recent years, metabolites derived from phytoalexins, such as glucoraphanin found in broccoli and other cruciferous vegetables (Brassicaceae), have gained much attention as potential cancer chemopreventive agents. The protective effect of these micronutrients is assumed to be due to the inhibition of Phase-I carcinogen-bioactivating enzymes and/or induction of Phase-II detoxifying enzymes, an assumption that still remains uncertain. The protective effect of glucoraphanin is thought to be due to sulforaphane, an isothiocyanate metabolite produced from glucoraphanin by myrosinase. Here we show, in rat liver, that while glucoraphanin slightly induces Phase-II enzymes, it powerfully boosts Phase-I enzymes, including activators of polycyclic aromatic hydrocarbons (PAHs), nitrosamines and olefins. Induction of the cytochrome P450 (CYP) isoforms CYP1A1/2, CYP3A1/2 and CYP2E1 was confirmed by Western immunoblotting. CYP induction was paralleled by an increase in the corresponding mRNA levels. Concomitant with this Phase-I induction, we also found that glucoraphanin generated large amount of various reactive radical species, as determined by electron paramagnetic resonance (EPR) spectrometry coupled to a radical-probe technique. This suggests that long-term uncontrolled administration of glucoraphanin could actually pose a potential health hazard.

Induction of cytochrome P450, generation of oxidative stress and in vitro cell-transforming and DNA-damaging activities by glucoraphanin, the bioprecursor of the chemopreventive agent sulforaphane found in broccoli.

The reduced cancer risk that appears to be linked to a diet rich in fruits and vegetables has fueled the belief that regular intake of isolated phytochemicals could potentially prevent cancer. In recent years, the glucosinolate metabolites derived from cruciferous vegetables, such as the isothiocyanate sulforaphane in broccoli, have gained much attention as potential cancer chemopreventive agents. The protective effect of sulforaphane, which is liberated from its glucosinolate precursor glucoraphanin (GRP) by myrosinase hydrolysis, is conventionally thought to involve the induction of Phase-II metabolizing enzymes. These Phase-II enzymes are implicated in the detoxication of many carcinogens and reactive oxygen species (ROS), thereby protecting cells against DNA damage and subsequent malignant transformation. While the induction of Phase-II enzymes is usually considered beneficial, in some cases these enzymes also bioactivate several hazardous chemicals. Furthermore, despite its projected benefits, the unknown effect of sulforaphane on Phase-I enzyme systems, which are involved in the bioactivation of a variety of carcinogens, should not be overlooked. Here we show that, in rat lungs, while GRP, the bioprecursor of the chemopreventive agent sulforaphane, slightly induced Phase-II detoxifying enzymes, it powerfully induced Phase-I carcinogen-activating enzymes, including activators of carcinogenic polycyclic aromatic hydrocarbons (PAHs). Concomitant with this Phase-I induction, GRP also over-generated ROS. Additionally, in a cell-transforming assay, GRP facilitated the metabolic activation of the PAH benzo[a]pyrene to reactive carcinogenic forms and in a yeast genotoxicity test it damaged DNA. This suggests that regular administration of GRP could actually increase rather than decrease cancer risk, especially in individuals exposed to environmental mutagens and carcinogens such as those found in tobacco smoke and in certain industrial settings.

Cell-cycle deregulation in BALB/c 3T3 cells transformed by 1,2-dibromoethane and folpet pesticides.

The cell-transforming potential of 1,2-dibromoethane and folpet, two widely used agricultural pesticides that are potential sources of environmental pollution, has been previously ascribed to their promoting activity. In this study, we investigated whether BALB/c 3T3 transformation by these chemicals was associated with the deregulation of signals involved in cell-cycle progression and in cell-cycle checkpoint induction. We found that two BALB/c 3T3 cell clones transformed by in vitro medium-term (8-week) exposure to the carcinogens had a constitutive acceleration of cell transition from G(1) to S phase and an abrogation of the radiation-induced G(1)/S checkpoint. These events involved multiple signals; in particular, the inhibitors of cyclin/cyclin-dependent kinase complexes p21 and p27 were significantly down-modulated and the positive regulators of cell-cycle progression cyclin D(3) and E were up-modulated. As anticipated for cells where the G(1)/S checkpoint was abrogated, the transformed cells exhibited a significant reinforcement of the radiation-induced G(2)/M checkpoint, the only checkpoint remaining to protect genomic integrity. However, cyclin A(1) and B(1) coexpression and cyclin A(1) overexpression were found despite the G2 arrest in irradiated cells and these signals likely attenuate the G(2)/M checkpoint. These alterations to normal cell cycling may promote the emergence of both numerical and structural chromosomal abnormalities and their tolerance. Such a condition could play a key role in neoplastic transformation and be crucial in tumor progression. Furthermore, cyclin A(1) overexpression may play an autonomous role in the neoplastic transformation of BALB/c 3T3 cells, as it does in other cell types of mesenchymal origin.

Beta-carotene: a cancer chemopreventive agent or a co-carcinogen?

Evidence from both epidemiological and experimental observations have fueled the belief that the high consumption of fruits and vegetables rich in carotenoids may help prevent cancer and heart disease in humans. Because of its well-documented antioxidant and antigenotoxic properties, the carotenoid beta-carotene (betaCT) gained most of the attention in the early 1980s and became one of the most extensively studied cancer chemopreventive agents in population-based trials supported by the National Cancer Institute. However, the results of three randomized lung cancer chemoprevention trials on betaCT supplementation unexpectedly contradicted the large body of epidemiological evidence relating to the potential benefits of dietary carotenoids. Not only did betaCT show no benefit, it was associated with significant increases in lung cancer incidence, cardiovascular diseases, and total mortality. These findings aroused widespread scientific debate that is still ongoing. It also raised the suspicion that betaCT may even possess co-carcinogenic properties. In this review, we summarize the current data on the co-carcinogenic properties of betaCT that is attributed to its role in the induction of carcinogen metabolizing enzymes and the over-generation of oxidative stress. The data presented provide convincing evidence of the harmful properties of this compound if given alone to smokers, or to individuals exposed to environmental carcinogens, as a micronutrient supplement. This has now been directly verified in a medium-term cancer transformation bioassay. In the context of public health policies, while the benefits of a diet rich in a variety of fruits and vegetables should continue to be emphasized, the data presented here point to the need for consideration of the possible detrimental effects of certain isolated dietary supplements, before mass cancer chemoprevention clinical trials are conducted on human subjects. This is especially important for genetically predisposed individuals who are environmentally or occupationally exposed to mutagens and carcinogens, such as those found in tobacco smoke and in industrial settings.

Ribosome-inactivating proteins depurinate poly(ADP-ribosyl)ated poly(ADP-ribose) polymerase and have transforming activity for 3T3 fibroblasts.

It has been known that ribosome-inactivating proteins (RIPs) from plants damage ribosomes by removing adenine from a precise position of rRNA. Subsequently it was observed that all tested RIPs depurinate DNA, and some of them also non-ribosomal RNAs and poly(A), hence the denomination of adenine polynucleotide glycosylases was proposed. We report now that ricin, saporin-L2, saporin-S6, gelonin and momordin depurinate also poly(ADP-ribosyl)ated poly(ADP-ribose) polymerase (auto modified enzyme), an enzyme involved in DNA repair. We observed also that all RIPs but gelonin induce transformation of fibroblasts, possibly as a consequence of damage to DNA and of the altered DNA repair system.

In vitro induction of benzo(a)pyrene cell-transforming activity by the glucosinolate gluconasturtiin found in cruciferous vegetables.

Cytotoxic and cell-transforming activity of gluconasturtiin (GNST), a promising chemopreventive agent commonly found in human diet, was studied in a medium-term bioassay utilizing BALB/c 3T3 cells. We also assessed whether GNST coupled with myrosinase, thus yielding product phenylethyl isothiocyanate (as shown by gas chromatography-mass spectral analysis), can affect the transforming potential of benzo(a)pyrene (B(a)P). Neither cytotoxicity nor cell-transforming activity was recorded. On the contrary, a marked increase (up to sevenfold) of the transforming activity of B(a)P was seen. This cocarcinogenic potential could be ascribed to an imbalance among bioactivation/detoxication during cell growth. These results indicate the need for an overall toxicological characterization of a chemopreventive agent prior to large-scale use.