Gian Franco Pedulli

Cytotoxic and antioxidant activity of 4-methylthio-3-butenyl isothiocyanate from Raphanus sativus L. (Kaiware Daikon) sprouts.

There is high current interest in the chemopreventive potential of Brassica vegetables (cruciferae), particularly due to their content in glucosinolates (GL), which upon myrosinase hydrolysis release the corresponding isythiocyanates (ITC). Some ITCs, such as sulforaphane (SFN) from broccoli ( Brassica oleacea italica), have been found to possess anticancer activity through induction of apoptosis in selected cell lines, as well as indirect antioxidant activity through induction of phase II detoxifying enzymes. Japanese daikon ( Raphanus sativus L.) is possibly the vegetable with the highest per capita consumption within the Brassicaceae family. Thanks to a recently improved gram scale production process, it was possible to prepare sufficient amounts of the GL glucoraphasatin (GRH) as well as the corresponding ITC 4-methylthio-3-butenyl isothiocyanate (GRH-ITC) from its sprouts. This paper reports a study on the cytotoxic and apoptotic activities of GRH-ITC compared with the oxidized counterpart 4-methylsulfinyl-3-butenyl isothiocyanate (GRE-ITC) on three human colon carcinoma cell lines (LoVo, HCT-116, and HT-29) together with a detailed kinetic investigation of the direct antioxidant/radical scavenging ability of GRH and GRH-ITC. Both GRH-ITC and GRE-ITC reduced cell proliferation in a dose-dependent manner and induced apoptosis in the three cancer cell lines. The compounds significantly ( p < 0.05) increased Bax and decreased Bcl2 protein expression, as well as producing caspase-9 and PARP-1 cleavage after 3 days of exposure in the three cancer cell lines. GRH-ITC treatment was shown to have no toxicity with regard to normal human lymphocytes (-15 +/- 5%) in comparison with SFN (complete growth inhibition). GRH and GRH-ITC were able to quench the 2,2-diphenyl-1-picrylhydrazyl radical, with second-order rate constants of 14.0 +/- 2.8 and 43.1 +/- 9.5 M(-1) s(-1), respectively (at 298 K in methanol), whereas the corresponding value measured here for the reference antioxidant alpha-tocopherol was 425 +/- 40 M (-1) s (-1). GRH reacted with H2O2 and tert-butyl hydroperoxide in water (pH 7.4) at 37 degrees C, with rate constants of 1.9 +/- 0.3 x 10(-2) and 9.5 +/- 0.3 x 10(-4) M(-1) s (-1) (paralleling recently developed synthetic antioxidants) being quantitatively (>97%) converted to GRE. It is demonstrated that GRH-ITC has interesting antioxidant/radical scavenging properties, associated with a selective cytotoxic/apoptotic activity toward three human colon carcinoma cell lines, and very limited toxicity on normal human T-lymphocytes.

β-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.

Measurement of oxidative stress by EPR radical-probe technique.

An EPR method for the measurement of the oxidative stress status in biological systems is described. The method is based on the X-band EPR detection of a persistent nitroxide generated under physiological or pseudo-physiological conditions by oxidation of a highly lipophylic hydroxylamine probe. The probe employed is bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)-decandioate which is administrated as hydrochloride salt. This probe is able to give a fast reaction with the majority of radical species involved in the oxidative stress. Furthermore, it crosses cell membranes and distributes in a biological environment without the need to alter or destroy compartmentation. The method is therefore suitable for quantitative measurements of ROS and can be applied to human tissues in real clinical settings. It has been successfully employed in systems of growing complexity and interest, ranging from subcellular fractions to whole animals and human liver.

Solvent and pH effects on the antioxidant activity of caffeic and other phenolic acids.

The antioxidant activity of several phenolic acids and esters has been investigated both in organic solutions and in large unilamellar phosphatidylcholine vesicles. In solution these compounds behaved as good antioxidants, with the exception of protocatechuic acid, due to the presence of the catechol moiety. Because their antioxidant activity followed an inverse dependence on the magnitude of their O-H bond dissociation enthalpies (BDE), the key mechanism of the chain-breaking action was attributed to hydrogen atom transfer (HAT) from the phenolic OH to peroxyl radicals. In unilamellar vesicles the antioxidant activity was strongly dependent on the pH of the buffer solution. In acid media (pH 4) all of the examined phenolic acids or esters behaved as weak inhibitors of peroxidation, whereas, with increasing pH, their antioxidant activity increased substantially, becoming comparable to or even better than that of Trolox. At pH 8 they also gave rise to lag phases 2-3 times longer than that of Trolox. The increased activity being observed in proximity of the pK(a) value corresponding to the ionization of one of the catecholic hydroxyl groups, this effect has been attributed to the high antioxidant activity of the phenolate anion.

The nature of prooxidant activity of vitamin C.

It was recently reported that vitamin C (500 mg/day for 6 weeks) administered as a dietary supplement to healthy humans exhibits a prooxidant, as well as an antioxidant effect in vivo. Here we show that high intakes of vitamin C (500 mg/kg b.w. for 4 days) in the rat are able to markedly induce hepatic cytochrome P4502E1-linked monooxygenases, measured as p-nitrophenol hydroxylase activity and corroborated by means of Western blot analyses. Furthermore, using Electron Paramagnetic Resonance Spectroscopy (EPR) coupled to a spin-trapping technique, we have also found that this induction generates large amounts of the anion radical superoxide (O2-). Therefore we can conclude that the adverse prooxidant outcomes (i.e. oxidative DNA damage) associated to vitamin C supplementation, being associated to a typical reversible boosting effect (i.e. enzymatic induction), may be easily controlled by a discontinuous supply. However, since the induced P4502E1 isoforms by vitamin C are responsible for ethanol metabolism to highly reactive radicals, care should be taken even in moderate drinkers.

Transition metal salts catalysis in the aerobic oxidation of organic compounds. Thermochemical and kinetic aspects and new synthetic developments in the presence of N-hydroxy-derivative catalysts

Abstract The catalytic system formed by Mn(II) and Co(II) or Cu(II) nitrates has shown to be particularly effective for the oxidation of ketones and aldehydes by molecular oxygen under mild conditions. The process has a general character, but it is particularly selective for the oxidation of alkyl–aryl ketones to aromatic carboxylic acids, alkyl-cyclopropyl ketones to cyclopropane carboxylic acids and cycloalkanones to dicarboxylic acids. Mn salt plays a key role in this catalysis, which catalyses the enolisation of the carbonyl compound and initiates a free-radical redox chain with oxygen by an electron-transfer process. Co and Cu salts alone are inert, but they exalt the catalytic activity of the Mn salt, being more effective in the decomposition of the hydroperoxides. The same metal salt complexes, associated with TEMPO revealed particularly effective for the aerobic oxidation of primary and secondary alcohols to the corresponding aldehydes and ketones under mild conditions (with air or oxygen at room temperature and atmospheric pressure); the mechanism of the catalysis is discussed. Thermochemical and kinetic investigations by EPR spectroscopy, concerning N-hydroxy compounds, have allowed to evaluate the Bond Dissociation Enthalpies (BDE) of several OH bonds and the absolute rate constants for the formation of the phthalimide-N-oxyl (PINO) radical from N-hydroxyphthalimide (NHPI) and for the hydrogen abstraction from several CH bonds by the PINO radical. The thermochemical and kinetic results have allowed us to explain the opposite catalytic behaviour of the two nitroxyl radicals, TEMPO and PINO, the former being an inhibitor of free radical processes, the latter a promoter of free-radical chain, and to develop new selective processes concerning the aerobic oxidation of alcohols, amines, amides, silanes and the substitution of heteroaromatic bases.

The Unusual Reaction of Semiquinone Radicals with Molecular Oxygen

Hydroquinones (benzene-1,4-diols) are naturally occurring chain-breaking antioxidants, whose reactions with peroxyl radicals yield 1,4-semiquinone radicals. Unlike the 1,2-semiquinone radicals derived from catechols (benzene-1,2-diols), the 1,4-semiquinone radicals do not always trap another peroxyl radical, and instead the stoichiometric factor of hydroquinones varies widely between 0 and 2 as a function of ring-substitution and reaction conditions. This variable antioxidant behavior has been attributed to the competing reaction of the 1,4-semiquinone radical with molecular oxygen. Herein we report the results of experiments and theoretical calculations focused on understanding this key reaction. Our experiments, which include detailed kinetic and mechanistic investigations by laser flash photolysis and inhibited autoxidation studies, and our theoretical calculations, which include detailed studies of the reactions of both 1,4-semiquinones and 1,2-semiquinones with O2, provide many important insights. They show that the reaction of O2 with 2,5-di-tert-butyl-1,4-semiquinone radical (used as model compound) has a rate constant of 2.4 +/- 0.9 x 10(5) M-1 s-1 in acetonitrile and as high as 2.0 +/- 0.9 x 10(6) M-1 s-1 in chlorobenzene, i.e., similar to that previously reported in water at pH approximately 7. These results, considered alongside our theoretical calculations, suggest that the reaction occurs by an unusual hydrogen atom abstraction mechanism, taking place in a two-step process consisting first of addition of O2 to the semiquinone radical and second an intramolecular H-atom transfer concerted with elimination of hydroperoxyl to yield the quinone. This reaction appears to be much more facile for 1,4-semiquinones than for their 1,2-isomers.

Catalytic Chain-Breaking Pyridinol Antioxidants

The synthesis of 3-pyridinols carrying alkyltelluro, alkylseleno, and alkylthio groups is described together with a detailed kinetic, thermodynamic, and mechanistic study of their antioxidant activity. When assayed for their capacity to inhibit azo-initiated peroxidation of linoleic acid in a water/chlorobenzene two-phase system, tellurium-containing 3-pyridinols were readily regenerable by N-acetylcysteine contained in the aqueous phase. The best inhibitors quenched peroxyl radicals more efficiently than alpha-tocopherol, and the duration of inhibition was limited only by the availability of the thiol reducing agent. In homogeneous phase, inhibition of styrene autoxidation absolute rate constants k(inh) for quenching of peroxyl radical were as large as 1 x 10(7) M(-1) s(-1), thus outperforming the best phenolic antioxidants including alpha-tocopherol. Tellurium-containing 3-pyridinols could be quantitatively regenerated in homogeneous phase by N-tert-butoxycarbonyl cysteine methyl ester, a lipid-soluble analogue of N-acetylcysteine. In the presence of an excess of the thiol, a catalytic mode of action was observed, similar to the one in the two-phase system. Overall, compounds bearing the alkyltelluro moiety ortho to the OH group were much more effective antioxidants than the corresponding para isomers. The origin of the high reactivity of these compounds was explored using pulse-radiolysis thermodynamic measurements, and a mechanism for their unusual antioxidant activity was proposed. The tellurium-containing 3-pyridinols were also found to catalyze reduction of hydrogen peroxide in the presence of thiol reducing agents, thereby acting as multifunctional (preventive and chain-breaking) catalytic antioxidants.

Free radical intermediates in the inhibition of the autoxidation reaction.

The autoxidation of organic materials is a detrimental radical chain process often leading to their rapid deterioration unless they are protected by preventive and/or chain breaking antioxidants. The properties of the more important family of the latter ones, that one of phenols, are illustrated in this tutorial review. A short outline of diarylamine antioxidants is also given. We describe simple experimental methods employed for the determination of the two parameters more useful for estimating the inhibiting power of antioxidants, that is the kinetic rate constant for their reaction with the chain carrying peroxyl radicals, kinh, to give persistent phenoxyl or aminyl radicals and the bond dissociation enthalpy BDE(X–H) (X = O, N) of the bond cleaved in the inhibition process. The dependence of these parameters on the number and nature of the substituents is discussed and, in the case of phenols, a simple rule allowing to predict with reasonable accuracy the BDE and kinh values, from their structure. The effect of solvent polarity on the antioxidant power is also described. Finally, the information on the mechanism of reaction between phenols and peroxyl radicals provided by both experiments and theoretical calculations are examined. Because of difficulties associated with the analysis of non-homogenous systems all the reported results refer to homogenous solution in which experimental data can be analysed by means of more reliable and complete treatments.

Dynamic aspects of cyclodextrin host-guest inclusion as studied by an EPR spin-probe technique

Major changes in the EPR parameters of benzyl tert-butyl nitroxide and other dialkyl nitroxides on complexation by cyclodextrins (CDs) make these radicals particularly suitable probes for studying inclusion phenomena, because the signals for the free and included species are well separated. Furthermore, selective line broadening of the EPR spectra enables the kinetics of inclusion to be investigated [Eq. (a)].