Roberto Pizzala

Anthocyanins protect against DNA damage induced by tert-butyl-hydroperoxide in rat smooth muscle and hepatoma cells.

Anthocyanins are flavonoids present in a variety of pigmented food and, like other flavonoids, seem to play a role in preventing human pathologies related to oxidative stress. In fact, anthocyanins have been shown to exert antiproliferative effects in cell cultures and exhibit antiinflammatory and vasoprotective activities in animal models. Although these biological activities have been related to their antioxidant properties, little is known on the molecular mechanism of action of anthocyanins. The effects of pretreatment with the anthocyanins delphinidin, cyanidin, and their glycoside and rutinoside derivatives against induction of DNA damage induced by tert-butyl-hydroperoxide (TBHP) were evaluated in rat smooth muscle and in rat hepatoma cell lines using alkaline single cell gel electrophoresis (Comet test). In addition, a possible protection exerted by anthocyanins on cell killing, lipid peroxidation, and redox state alterations induced by TBHP was also investigated. It was found that the treatment with TBHP induces the formation of DNA single strand breaks (SSB) and oxidised bases, along with cell killing, lipid peroxidation and redox state alteration. Our data demonstrate that anthocyanins are effective against cytotoxicity, DNA SSB formation and lipid peroxidation induced by TBHP, but they do not have any detectable effect against impairment by TBHP of cellular redox state and on protection against DNA bases oxidation. The presence of a sugar moiety in anthocyanin derivatives reduced this protective effect, mainly in rat hepatoma cells. The different activity of anthocyanins and their derivatives may be explained taking into account a structure/function relationship that could also influence anthocyanin intracellular localisation.

Grape waste extract obtained by supercritical fluid extraction contains bioactive antioxidant molecules and induces antiproliferative effects in human colon adenocarcinoma cells.

Grape waste management is one of the main problems of winery industries, but, conversely, grape waste contains a high amount of polyphenols that might protect against human diseases related to oxidative stress, such as colorectal cancer. Therefore, the aim of this work was to investigate the antioxidant and antiproliferative activities of a grape waste extract obtained by supercritical fluid extraction. Because the beneficial effect of grape is related to its content of polyphenolic molecules, the extract was chemically characterized by high-performance liquid chromatography in order to assess its major bioactive components. The antioxidant activity of the grape extract was determined. The results showed that the grape extract presents a strong antiradical activity in the in vitro 2,2-diphenyl-1-picrylhydrazyl radical assay and protects against reactive oxygen species production in human colon adenocarcinoma cells (Caco-2). In contrast, the extract did not protect in the citronellal thermooxidation system and showed a weak protective action against lipid peroxidation in Caco-2 cells. The clonogenic assay and the cell cycle distribution analysis showed that the grape extract has a significant antiproliferative effect in a tumor cell line. These data indicate that grape extract is a promising product to be used as an anti-free radical agent and could exert a chemopreventive action.

Effects of β-carotene and α-tocopherol on photogenotoxicity induced by 8-methoxypsoralen: The role of oxygen

Abstract The protective effect of β-carotene (β-C) and α-tocopherol (α-T), singularly and in equimolar mixtures, toward the photomutagenicity induced by 8-methoxypsoralen (8-MOP), at different oxygen partial pressure (pO 2 ), was evaluated in two different experimental models: Salmonella typhimurium TA102 and Saccharomyces cerevisiae D7. After phototreatment with 8-MOP, the results show a lethal effect under hypoxic conditions in both experimental model systems, an increase in revertants associated to the pO 2 increase in S. typhimurium TA102, and a decrease in revertants and convertants associated to the pO 2 increase in S. cerevisiae D7. In S. typhimurium TA102, in atmospheric condition, β-C and α-T (1.86 or 18.6 μM) show a protective effect only at the higher dosage. α-T was more protective than β-C. The equimolar mixtures show an antimutagenic effect at both dosage used with a synergistic effect at lower dosage and an additive antimutagenic activity at higher dosage. An inhibition of the spontaneous mutagenicity by mixtures at higher dosage was also observed. The results obtained in S. typhimurium TA102 show an antimutagenic effects of β-C, α-T and their mixture at 190 mmHg pO 2 , confirming the data obtained in air condition. At 380 mmHg pO 2 , α-T and the mixture show a significant antimutagenic activity; at 570 mmHg pO 2 , only α-T is protective. At 760 mmHg pO 2 , no protective effect was observed by the two antioxidants, and β-C increases the photomutagenicity induced by 8-MOP. In S. cerevisiae D7 a protective effect was only observed at 380 mmHg pO 2 with the mixture. No antigenotoxic effect was found in the other experimental conditions, even if the uptake of the two antioxidants was confirmed by HPLC. Our results underline the role of oxygen in the photomutagenicity induced by 8-MOP and in the antimutagenic activity of β-C and α-T. This is the first report confirming in a cellular experimental model the data obtained in some chemical systems: the protective effect of β-C only at low pO 2 and the synergistic effect of mixture of β-C and α-T.

Genotoxicity assay of five pesticides and their mixtures in Saccharomyces cerevisiae D7

Four organophosphorus pesticides (azinphos-methyl, diazinone, dimethoate, and pirimiphos-methyl), and one carbamate (benomyl) were tested for cytotoxicity, reverse mutation and gene conversion in Saccharomyces cerevisiae D7, with and without the S9 metabolic system. Furthermore, two mixtures of the above compounds, namely benomyl + pirimiphos-methyl (6/1 ratio) and dimethoate + diazinone + azinphos-methyl (10/4/6 ratio) were tested in the same experimental model. Azinphos-methyl, benomyl, and pirimiphos-methyl alone did not induce any genotoxic effect, whereas azinphos-methyl and diazinone were active in inducing reversion and gene conversion. The benomyl + pirimiphos-methyl mixture did not show any genotoxic activity. The dimethoate + diazinone + azimphos-methyl mixture was genotoxic, although an antagonistic effect between the components was observed. The addition of S9 post-mitochondrial liver fraction decreased the activity of both single and mixed genotoxic agents.

Structure–activity relationship and role of oxygen in the potential antitumour activity of fluoroquinolones in human epithelial cancer cells

The photobehavior of ciprofloxacin, lomefloxacin and ofloxacin fluoroquinolones was investigated using several in vitro methods to assess their cytotoxic, antiproliferative, and genotoxic potential against two human cancer cell lines. We focused our attention on the possible relationship between their chemical structure, O₂ partial pressure and photobiological activity on cancer cells. The three molecules share the main features of most fluoroquinolones, a fluorine in 6 and a piperazino group in 7, but differ at the key position 8, unsubstituted in ciprofloxacin, a fluorine in lomefloxacin and an alkoxy group in ofloxacin. Studies in solution show that ofloxacin has a low photoreactivity; lomefloxacin reacts via aryl cation, ciprofloxacin reacts but not via the cation. In our experiments, ciprofloxacin and lomefloxacin showed a high and comparable potential for photodamaging cells and DNA. Lomefloxacin appeared the most efficient molecule in hypoxia, acting mainly against tumour cell proliferation and generating DNA plasmid photocleavage. Although our results do not directly provide evidence that a carbocation is involved in photodamage induced by lomefloxacin, our data strongly support this hypothesis. This may lead to new and more efficient anti-tumour drugs involving a cation in their mechanism of action. This latter acting independently of oxygen, can target hypoxic tumour tissue.

Lack of molecular relationships between lipid peroxidation and mitochondrial DNA single strand breaks in isolated rat hepatocytes and mitochondria.

We investigated the molecular relationships between lipid peroxidation and mitochondrial DNA (mtDNA) single strand breaks (ssb) in isolated rat hepatocytes and mitochondria exposed to tert-butylhydroperoxide (TBH). Our results show that mtDNA ssb induced by TBH are independent of lipid peroxidation and dependent on the presence of iron and of hydroxyl free radicals. These data contribute to the definition of the mechanisms whereby mtDNA ssb are induced and provide possible molecular targets for the prevention of this kind of damage in vivo.