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
Moreno Paolini1,
Paolo Perocco2,
Donatella Canistro1,
Luca Valgimigli3,
Gian Franco Pedulli3,
Renato Iori4,
Clara Della Croce5,
Giorgio Cantelli-Forti1,6,
Marvin S. Legator6 and
Sherif Z. Abdel-Rahman6,7
1 Department of Pharmacology, 2 Institute of Cancerology, 3 Department of Organic Chemistry A. Mangini, Alma-Mater StudiorumUniversity of Bologna, Bologna, Italy, 4 Research Institute for Industrial Crops (MIPAF), Bologna, Italy, 5 Institute of Biology and Agricultural Biotechnology (IBBA)CNR Research Area, Pisa, Italy and 6 Department of Preventive Medicine and Community Health, Division of Environmental Toxicology, The University of Texas Medical Branch at Galveston, Texas, USA
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Abstract
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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.
Abbreviations: EPR, electronic paramagnetic resonance; GRP, glucoraphanin; GST, glutathione S-transferase; PROD, pentoxyresorufin O-dealkylase; ROS, reactive oxygen species; SOD, superoxide dismutase; TH, testosterone hydroxylase; UDP-GT, UDP-glucuronosyl transferase
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Introduction
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Since the 1980 s, public health recommendations have emphasized the health benefits associated with the consumption of fresh fruits and vegetables (1). Epidemiological and animal studies linking the intake of a variety of plant foods with a major reduction in the incidence of lung cancer and other malignancies provide ample support for this policy (2). Beneficial effects are widely attributed to specific phytochemicals such as the isothiocyanate sulforaphane (3). However, sulforaphane is not present as such in plants but is derived via myrosinase hydrolysis from its thioglucoside precursor, glucoraphanin (GRP), which is predominantly found in cruciferous vegetables such as broccoli. This hydrolysis reaction occurs either directly, when raw vegetables are chopped or chewed, or indirectly, by intestinal microflora, when cooked vegetables are ingested (4).
The beneficial protective effect of sulforaphane, which is used physiologically by plants in defense against environmental stress or pathogens, is thought to involve a monofunctional induction of Phase-II enzymatic systems that are generally believed to detoxify carcinogens, mutagens and reactive oxygen species (ROS), thereby protecting cells against DNA damage and neoplastic transformation (5,6). In an earlier report (7), we pointed out the error in the concept that Phase-II metabolizing enzymes are always detoxifying, as these enzymes can also act as activators for some specific classes of chemicals. We have also warned that, despite its projected benefits, the unknown effect of broccoli-derived chemicals on other enzyme systems, such as the Phase-I enzyme systems that are involved in bioactivation of a variety of carcinogens, should not be overlooked. In the presence of exposure to certain environmental carcinogens, such as those found in tobacco smoke and in some industrial settings, this can result in the formation of reactive carcinogenic metabolites leading to increased cancer risk (8).
The synthetic isothiocyanate sulforaphane has been widely used in many investigations as a surrogate for the major natural constituents of cruciferous vegetables. However, this is not strictly correct, as this isothiocyanate is actually a secondary metabolite produced by enzymatic degradation of a natural constituent, the glucosinolate GRP (Figure 1).
Recently, we developed a method to produce the natural precursor of sulforaphane, GRP (9). In order to closely mimic the actual dietary exposure to sulforaphane following consumption of cruciferous vegetables, we have now utilized a rodent bioassay to examine the effects of GRP administration on both Phase-I bioactivating and Phase-II detoxifying enzymes, and on the production of ROS in rat lungs. Using a medium-term in vitro bioassay, we also investigated whether GRP could influence the carcinogenic potential of the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene, which is found in tobacco smoke and is an industrial pollutant. In addition, we studied the DNA-damaging potential of this glucosinolate on yeast cells.
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Materials and methods
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Chemicals
NADP+, NADPH, 7-ethoxyresorufin, corticosterone, testosterone and androst-4-ene-3,17-dione were purchased from Sigma Chemical Co. (St Louis, MO); glucose 6-phosphate and glucose 6-phosphate dehydrogenase from Boehringer-Mannheim (Germany); ethoxyresorufin, pentoxyresorufin and methoxyresorufin from Molecular Probes (Eugene, OR); HPLC grade methanol, tetrahydrofuran and dichloromethane from Labscan Co. (Dublin, Ireland); 16ß-hydroxytestosterone from Steraloids (Wilton, NH); 2
-, 2ß-hydroxytestosterone and echinenone were a generous gift from Dr P.G.Gervasi (CNR Pisa, Italy). The hydroxylamine probe bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate was synthesized in our laboratories following a previously described procedure and used as hydrochloride salt (10). All other chemicals and solvents used were of the highest purity commercially available.
Treatment of animals and preparation of subcellular fractions
Male SpragueDawley rats (Harlan-Nossan, Milan), weighing 150170 g, were housed under controlled conditions (12 h lightdark cycle, 22°C, 60% humidity). They were fed a rodent chow and had tap water ad libitum. GRP was synthesized through chemoselective oxidation of glucoerucin, which was isolated from the ripe seeds of Eruca sativa, as described previously (9). GRP was dissolved in saline and administered orally by gavage at 120 or 240 mg/kg body wt in a single dose or repeated doses (daily for four consecutive days). Control animals received the vehicle (saline) only. Ten rats were used in each group. Rats were killed by cervical dislocation after stunning by rotation, in accordance with approved Italian Ministerial procedures appropriate to the species. Animals were fasted for 16 h prior to killing, which occurred 24 h after the last treatment. They were killed humanely. Lungs were rapidly removed and processed separately, and the S9 fraction (9000 g) was then prepared (11). The post-mitochondrial supernatant was then centrifuged for 60 min at 105 000 g, the pellet was resuspended in 0.1 M K2P2O7, 1 mM EDTA (pH 7.4) and centrifuged again for 60 min at 105 000 g to give the final fraction. Washed microsomes were then resuspended with a hand-driven Potter Elvehjem homogenizer in a 10 mM TrisHCl buffer (pH 7.4) containing 1 mM EDTA and 20% (v/v) glycerol. Fractions were immediately frozen in liquid nitrogen and stored at -80°C until use.
Pentoxyresorufin O-dealkylase (PROD), ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities
The reaction mixture consisted of 0.025 mM MgCl2, 200 mM pentoxyresorufin, 0.32 mg of lung microsomal proteins and 130 mM NADPH in 2.0 ml 0.05 M TrisHCl buffer (pH 7.4). Resorufin formation at 37°C was calculated by comparing the rate of increase in relative fluorescence to the fluorescence of known amounts of resorufin (excitation 562 nm, emission 586 nm) (12). EROD and MROD activities were measured in the same manner as described for the pentoxyresorufin assay, except that the concentration of substrates was 1.7 mM ethoxyresorufin and 5 mM methoxyresorufin (13).
Testosterone hydroxylase (TH) activity
Incubation and isolation
Incubations contained lung microsomes (equivalent to 12 mg protein), 0.6 mM NADP+, 8 mM glucose 6-phosphate, 1.4 U glucose 6-phosphate dehydrogenase and 1 mM MgCl2, in a final volume of 2 ml 0.1 M phosphate Na+/K+ buffer (pH 7.4). The mixture was pre-incubated for 5 min at 37°C. The reaction was performed at 37°C by shaking, and started by the addition of 80 mM testosterone (dissolved in methanol). After 10 min, the reaction was stopped with 5 ml ice-cold dichloromethane and 12 nmol corticosterone (internal standard) in methanol. After 1 min vortexing, phases were separated by centrifugation at 2000 g for 10 min and the aqueous phase was extracted once more with 2 ml dichloromethane. The organic phase was extracted with 2 ml 0.02 N NaOH to remove lipid constituents, dried over anhydrous sodium sulfate and transferred to a small tube. Dichloromethane was evaporated at 37°C under nitrogen and the dried samples stored at -20°C. The samples were dissolved in 100 ml methanol and analysed by HPLC (14).
HPLC separation and quantification
Chromatographic separations were performed using a system consisting of a high-pressure pump (Waters Model 600E, Multisolvent Delivery System), a sample injection valve (Rheodyne Model 7121, Cotati, CA) with a 20 ml sample loop and an ultraviolet (UV) detector (254 nm, Waters Model 486, Tunable Absorbance Detector) connected to an integrator (Millennium 2010, Chromatography Manager). For reversed-phase separation of metabolites, a NOVA-PAK C18 analytical column (60 Å, 4 mm, 3.9 x 150 mm, Waters) was used as the stationary phase. The mobile phase consisted of a mixture of solvent A [7.5% (v/v) tetrahydrofuran in water] and solvent B [7.5% (v/v) tetrahydrofuran and 60% (v/v) methanol in water] at a 1 ml/min flow rate. Metabolite separation was performed by a gradient from 30 to 100% (v/v) of solvent B over 30 min. The eluent was monitored at 254 nm and the area under the absorption band was integrated. The concentration of metabolites was determined by the ratio between respective metabolite peak areas and corticosterone (internal standard) and in comparison with the calibration curves obtained with synthetic testosterone derivatives (15,16).
Determination of selected Phase-II enzyme activities
The incubation mixture for measuring glutathione S-transferase (GST) activity contained 1 mM glutathione, 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) in methanol and 0.025 ml of sample in a final volume of 2.5 ml 0.1 M phosphate Na+/K+ buffer (pH 6.5). The product of the reaction of the thiol group of glutathione with the electrophilic group of CDNB was read at 340 nm (
= 9.6/mM/cm) (17). UDP-glucuronosyl transferase (GT) was determined kinetically using 1-naphthol as substrate (final concentration, 50 mM) by the continuous fluorimetric (excitation l = 293, emission l = 335 nm) monitoring of 1-naphtholglucuronide production in the presence of 1 mM uridine-5'-diphosphoglucuronic acid (18). Experiments were performed in the presence or absence of Triton X-100 (0.2%) as detergent, in order to improve the assay sensitivity (19).
Electronic paramagnetic resonance (EPR) spin probe technique
Microsomes from the lungs of GRP-treated and control male animals were incubated at 37°C in standard EPR capillary tubes in 0.01 M Na+/K+ phosphate buffer (pH 7.4) in the presence of 1 mM NADPH and 0.5 mM of the hydroxylamine probe, bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate, synthesized as described previously (20). After mixing, the samples were immediately placed within the spectrometer's EPR cavity. The nitroxide radicals generated by reaction of the probe with the ROS present in the samples were then measured by EPR on a Bruker ESP 300 spectrometer equipped with an NMR gaussmeter for field calibration, a Bruker ER 033 M FF-lock and a Hewlett-Packard 5350B microwave frequency counter for the determination of the g-factor which was corrected with respect to that of perylene radical cation in concentrated H2SO4 (g = 2.00258). Spectra were recorded using the following instrumental settings: modulation amplitude = 1.0 G; conversion time = 163.84 ms; time constant = 163.84 ms; receiver gain 1.0 e 5; microwave power = 6.3 mW. The intensity of the first spectral line of the nitroxide was used to obtain the absolute amount of radicals per tissue milligram after calibration of the spectrometer response with a standard solution of TEMPO-choline in water, using an artificial ruby crystal as internal standard. In order to evaluate the extent of oxidation of the hydroxylamine by atmospheric oxygen under our experimental conditions, a reference sample containing only the hydroxylamine in physiologic solution was prepared for each sample and treated in the same way.
Medium-term cell-transforming bioassay
The BALB/c 3T3 medium-term cell-transforming bioassay was used to directly test the co-carcinogenic potential of GRP (21). BALB/c 3T3 cells (clone A31) were exposed to either 1.0 mg/ml GRP in the presence of 0.058 U myrosinase, 2.5 mg/ml benzo[a]pyrene alone (dissolved in 0.5% dimethylsulfoxide, DMSO), or GRP plus benzo[a]pyrene in the presence of myrosinase for 68 weeks in a level-II transformation test (21). Cells exposed to 1.0 mg/ml GRP in the absence of myrosinase served as controls. Twelve plates for each treatment were scored for the transformation frequency index as described earlier (22).
Genotoxicity tests in yeast cells
Experiments were performed as described previously (23). Briefly, the D7 strain of yeast Saccharomyces cerevisiae, obtained from F.K.Zimmermann, was used. Mitotic gene conversion and reverse point mutations were measured at the trp 5 and ilv1 loci, respectively (23). About 107 cells/ml were inoculated in liquid complete medium containing 2% glucose, 2% bactopeptone and 1% yeast extract and incubated at 30°C for 48 h up to the stationary phase (100200 x 106 cells/ml) in the presence of different concentrations of GRP. Cells were counted and plated, after suitable dilutions, on complete and selective media to ascertain survival convertants and revertants.
CYP content and protein concentration determination
Yeast cells harvested from the logarithmic phase were resuspended with a buffer containing 50 mM TrisHCl, 10 mM EDTA, 0.8 M Sorbitol, pH 7.4. CYP content was determined essentially according to the Omura and Sato method (24). Protein concentration was determined after diluting the samples 200 times, using the method described by Lowry et al. (25) and revised by Bailey (26), with bovine serum albumine utilized as a standard.
Statistical analysis
Statistical evaluation was performed using the Wilcoxon' rank test to assess significant differences in enzyme modulation between the groups of treated animals compared with control (27). The Student's t-test, corrected for the number of comparisons, was used to assess significant differences in the transformation frequency index between treatments in the BALB/c 3T3 cell-transforming bioassay, and in the mitotic frequencies of gene conversions and reverse point mutations in the yeast genotoxicity test.
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Results
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HPLC and fluorimetric analysis of Phase-I and Phase-II metabolizing enzymes in lungs of rats supplemented with GRP
We investigated the effects of administration of GRP, the precursor of the isothiocyanate sulforaphane, on the Phase-I microsomal mixed function monooxygenases in rat lungs. The results presented in Table I indicate that, with the exception of the single low dose (120 mg/kg body wt), the CYP1A1-linked EROD activity was significantly (P < 0.01) induced (up to 14-fold with repeated administration of 240 mg/kg body wt). A similar response was observed for the CYP1A2-linked MROD monooxygenases, which were induced up to 4.7-fold. A modest, but significant increase (P < 0.05) of PROD activity (CYP2B1/2) was observed with a single GRP dose of 240 mg/kg body wt. This increase was more pronounced in animals receiving repeated doses of the compound, reaching a 7.5-fold increase with a GRP dose of 240 mg/kg body wt. Among the stereo- and regio-selection hydroxylations of testosterone, the 16ß-(CYP2B1, CYP2C11)-TH activity was induced by GRP administration up to 4.3-fold by the repeated high dosage of GRP, while it was only slightly affected by the single treatments. 2ß-(CYP3A1/2-CYP1A1)-TH activity was significantly induced following either single or repeated doses of GRP up to 11.5-fold at 240 mg/kg body wt (repeated, P < 0.01). 2
-(CYP2C11)-TH activity was appreciably induced in the repeated treatment only, with a 8.5-fold increase observed at the highest dosage. Finally, 4-androsten-3,17-dione-(CYP3A1)-supported oxidase activity was also affected (
2-fold increase, high dosage) following repeated doses of GRP (Table I).
Markers of Phase-II metabolism, GST and UDP-GT activities were also evaluated in this study. Table II reports the expression of both GST and UDP-GT activities in lungs of rats supplemented with GRP. GST activity, measured using p-chloronitrobenzene as the substrate, was slightly induced in the repeated treatments only (up to 1.6-fold at 240 mg/kg body wt daily dose). In contrast, UDP-GT activity, determined using 1-naphthol as substrate, either in the presence or absence of TX-100, remained unaffected in all the experimental situations.
EPR measurements
Samples prepared by incubating
1 mg of lung microsomal proteins for 110 min at 37°C in 0.01 M Na+/K+ phosphate buffer (pH 7.4) containing 1 mM NADPH and 0.5 mM hydroxylamine probe was analysed by EPR spectroscopy. An intense three-line EPR spectrum attributed to the corresponding nitroxide on the basis of its spectral parameters (an = 15.524, g = 2.0062) was observed. We found a significant correlation between the induction of CYP content in subcellular lung preparations and the over-generation of ROS (r2 = 0.99, P = 0.006, Figure 2). Experiments performed in the presence of SOD (1000 U/mg) or SOD and catalase (for each 1000 U/mg) show that the formation of the nitroxide was strongly impaired, indicating that superoxide is mainly responsible for the oxidation of the hydroxylamine probe. Thus, under our experimental conditions, these enzymes reduced the amount of nitroxide detected to
10% of the original value. However, when the microincubations were performed in the presence of SOD, which had been denatured previously by thermal shock, the EPR signal that was recorded ranged from 15 to 25% of that achieved in the absence of the enzyme under the same conditions. This indicates that the role of this enzyme consists mostly of a general non-specific radical scavenging activity, presumably due to the many -SH groups present in the protein itself (data not shown).

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Fig. 2. Oxygen free-radical production by GRP. The three-line spectrum (an = 15.05, g = 2.0062) of stable nitroxide was recorded at regular intervals. R is the intensity of the nitroxide EPR signal.
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In vitro effects of GRP on cell transformation, DNA damage and CYP expression
The cell-transforming bioassay involving BALB/c 3T3 cells was used to directly test the co-carcinogenic potential of GRP and the environmental carcinogen benzo[a]pyrene selected as a test compound. We found that GRP significantly (P < 0.0001) enhances the conversion of benzo[a]pyrene to its ultimate carcinogenic forms. This was documented by the
6-fold increase in the transformation frequency index observed between cultures treated with benzo[a]pyrene alone and cultures treated with benzo[a]pyrene and GRP (in the presence of myrosinase) (Figure 3).

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Fig. 3. Cell-transforming activity of GRP. Data are mean values ± standard deviation referred to as transformation frequency index, calculated from transformation foci in 12 replicates and surviving cells. **P < 0.0001, treated cells versus control (Student's t-test).
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To further document the DNA-damaging potential of GRP, in the current study we used a short-term in vitro genotoxicity bioassay involving S.cerevisiae yeast cells. The results presented in Table III indicate that in stationary-phase growing cells, GRP at three concentrations tested (10, 100 and 1000 µM) did not affect point mutation frequency. However, in the presence of myrosinase, it significantly (P < 0.05) induced gene conversion frequency (up to
4-fold) compared with controls at the 10 mM concentration (the lowest concentration tested). Gene conversion was also significantly induced at 100 mM GRP, but the extent of induction was lower than that observed with the lower concentration (
2.6-fold), probably due to the cytotoxic effect.
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Table III. Effects of GRP on survival (surv), mitotic gene conversion (conv) and reverse point mutation (rev) in the diploid D7 strain of yeast S.cerevisiae
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We also determined in vitro CYP induction with GRP alone, particularly at the 1 mM concentration (P < 0.01), and with GRP in the presence of the enzyme myrosinase at either concentration (1 and 100 mM). The results presented in Table IV indicate that when yeast cells, harvested during logarithmic phase after exposure to GRP for 6 h, were resuspended in 50 mM HCl buffer, there was a significant increase in CYP measured in whole GRP-exposed cells compared with controls.
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Discussion
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We report here for the first time the effects on Phase-I and Phase-II xenobiotic metabolizing enzymes in lung microsomes of single and repeated administration of GRP to rats. The doses that we used in this investigation are comparable with the doses that have shown chemopreventive effects in animal studies (5). Although glucosinolate content in cruciferous vegetables such as broccoli varies according to the species, cultivation, and the parts of the plant used, the mean content is
100 mmol/100 g fresh weight (a typical portion of vegetables), corresponding to 26 mg GRP (
55% of the total). Individual specific substrates to different CYPs and to testosterone (as a multi-bioprobe) were used for oxidative reactions. Conjugating reactions were also studied using selected probes. In addition, EPR spectroscopy, coupled with a spin-trapping technique, was employed to measure the generation of ROS by GRP. Using in vitro models, we also investigated the transforming and DNA-damaging potential of GRP. To confirm Phase-I modulation resulting from exposure to GRP, cultures of yeast cells were also used to study CYP up-regulation by GRP.
A powerful and highly significant increase (
4.413-fold) of several Phase-I carcinogen-metabolizing enzymes in rat lung microsomes was detected following a single or repeated treatments of GRP. We found a significant increase in the following CYP enzymes: CYP1A1/2 (which activate polychlorinated biphenyls, aromatic amines and PAHs), CYP3A1/2 (which activate nitropyrenes, aflatoxins and PAHs), CYP2B1/2 (which activate olefins and halogenated hydrocarbons) and CYP2C11 (which activates nitrosamines, aflatoxins and ochratoxins). This increase in Phase-I enzymes was documented using the following substrates as probes for different CYP isoenzymes: ethoxyresorufin (CYP1A1), methoxyresorufin (CYP1A2), penthoxyresorufin O-dealkylase (CYP2B1/2), testosterone 16ß-hydroxylase (CYP2B1, CYP2C11), 16ß-hydroxylation (CYP2B1, CYP2C11), 2ß-hydroxylation (CYP1A1 and CYP3A1), 2
-hydroxylation (CYP2C11).
The observed CYP induction following GRP administration suggests that GRP may possess co-carcinogenic properties. This may be of particular concern to heavy smokers who are exposed to a wide range of tobacco procarcinogens that are activated by these CYP enzyme systems. There are over 4000 chemicals in tobacco smoke, among which at least 40 have been identified as carcinogens, as tumor initiators, or as promoters in laboratory animals (28). Indeed, if extrapolated to humans, similar increases in CYP levels could pose a significant risk of lung cancer to heavy smokers. Moreover, as many of these tobacco smoke procarcinogens are themselves CYP inducers (28) (e.g. PAHs that induce CYP1A1/2), they could act synergistically with GRP to impose an even greater cancer risk. This phenomenon could be intensified further in genetically predisposed individuals who inherit certain high-risk genotypes affecting carcinogen-metabolizing enzymes (29). Induction and/or polymorphisms of several CYP enzymes have been widely linked with increased risk of lung cancer (29,30). The risk may be even higher in individuals who lack adequate detoxication mechanisms or who have reduced DNA repair capacity due to polymorphisms in genes of detoxifying enzymes or DNA repair enzymes (2934).
Because of the widely recognized role of ROS as a factor that increases the risk for development of cancer in humans (28), we used electron EPR spectroscopy coupled to a spin-trapping technique to evaluate the precise contribution of GRP-induced CYP on free radical generation (11,20). We found a notable correlation between CYP up-regulation in rat lungs and the production of ROS. The latter could act synergistically with other ROS present in cigarette smoke, such as nitrogen dioxide, peroxyl radicals and hydroquinones, to impose even greater oxidative stress in the lungs of smokers (28), and hence a greater cancer risk.
We used the medium-term cell-transforming bioassay involving BALB/c 3T3 cells to directly test the co-carcinogenic potential of GRP. This in vitro model correlates well (7085%) with in vivo long-term carcinogenicity bioassays (21). We found that GRP remarkably enhances the conversion of benzo[a]pyrene, selected as a model test compound, to its ultimate carcinogenic metabolites. This increase in cell-transforming activity is consistent with the observed inducing effect of GRP toward CYP1A1, the main enzyme responsible for benzo[a]pyrene bioactivation. It can be argued that by inducing Phase-I CYP bioactivating enzymes, GRP may trigger the conversion of benzo[a]pyrene to carcinogenic reactive intermediates such as diol-epoxides. This argument is supported by recent studies showing that a cruciferae-based diet containing a selection of brassicaceous vegetables, including broccoli, Brussel sprouts or cauliflower, leads to a significant increase in Phase-I enzymes such as CYP1A1/2. These enzymes can bioactivate PAHs, dioxins, aromatic amines and nitrosamines (35,36). Further support for this effect is provided by a recent population study. This randomized intervention study documented an increase in CYP1A2 activity when individuals consumed a controlled diet containing a selection of cruciferous vegetables (37). This study also showed a decrease in Phase-I enzyme activity when subjects consumed diets containing a selection of apiacieous vegetables, such as celery, dill weed and parsley. The apparent enhancement of the cell-transforming effect of benzo[a]pyrene by GRP may be particularly important for those genetically predisposed individuals who possess specific polymorphisms in the bioactivation and detoxication enzymes for procarcinogens, such as benzo[a]pyrene (29,30,39), or who possess polymorphisms in the DNA repair genes responsible for the repair of the resulting genetic damage (3234).
With an additional in vitro short-term bioassay using yeast cells as a biologic test system (the D7 strain of S.cerevisiae), we have further corroborated our earlier results. We found that GRP markedly affects mitotic gene conversion, an end-point indicating DNA damage. It is noteworthy to mention that some toxicological effects of brassicaceous vegetables have also been demonstrated in other in vitro investigations (38). In particular, the genotoxic activity of crude juices from Brassica vegetables, extracts from cruciferous-derived phytopharmaceutical preparations, and spices originating from cruciferous plants have been reported.
In summary, we present novel findings indicating that, rather than behaving as a chemopreventive agent, GRP could actually exert toxicological effects by inducing carcinogen-bioactivating enzymes and by generating oxidative stress. Our data show that GRP is also able to damage DNA. The boost observed in Phase-I enzymes may be of special concern for genetically susceptible individuals who are environmentally and/or occupationally exposed to mutagens and carcinogens known to be metabolized by Phase-I bioactivating enzymes. In the context of public health control policies, while the benefits of a diet rich in a variety of fruits and vegetables should continue to be emphasized, our study points to the need for consideration of the possible detrimental effects of certain isolated cruciferous-derived dietary supplements before mass cancer chemoprevention clinical trials are designed and conducted. Considering that sulforaphane, like many other isothiocynates, has been found to induce cell cycle arrest and apoptosis, this supplement might be a better candidate for chemotherapy rather than for chemoprevention (4345). Our study provides a further example of the pitfalls (40,41) that can be encountered if simplistic attempts are made to reproduce the benefits of a varied plant-based diet (containing thousands of substances in a natural matrix) by means of extensive consumption of a single type of plant, or mass administration of specific isolated phytochemicals (42).
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Notes
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7 To whom correspondence should be addressed Email: sabdelra{at}utmb.edu 
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Acknowledgments
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We are grateful to Robin M.T.Cooke for scientific editing and to Dr Marinel Ammenheuser for editorial assistance. This work was supported by the Italian Ministero dell'Istruzione, Università e Ricerca (MIUR) and, in part, by Philip Morris Inc.
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Received February 28, 2003;
revised September 8, 2003;
accepted September 10, 2003.