Resveratrol and its methoxy-derivatives as modulators of DNA damage induced by ionising radiation.

Various naturally occurring stilbene-like compounds that are related to resveratrol (RSV) possess some of the beneficial effects of the parent molecule and provide even further benefits. Therefore, a series of methoxylated analogues of RSV were prepared with the aim of increasing antitumour and proapoptotic activity. In a previous article, we studied two methoxy-derivatives, pterostilbene (PTERO) and trimethoxystilbene (TRIMETHOXY), in which the first was formed by the substitution of two hydroxyl groups with two methoxy groups (trans-3,5-dimethoxy-4'-hydroxystilbene) and the second was formed by the replacement of all three OH groups with methoxy groups (trans-3,5,4'-trimethoxystilbene). Both methoxy-derivatives showed stronger antioxidant activity when compared with RSV. In the present article, we focused on the analysis of the ability of RSV and its two methoxylated derivatives to protect proliferating non-tumoural cells from the damage induced by ionising radiation (IR). First we showed that the methoxy derivatives, contrary to their parental compound, are unable to affect topoisomerase enzyme and consequently are not clastogenic per se Second we showed that both PTERO and TRIMETHOXY more efficiently reduce the chromosome damage induced by IR. Furthermore, TRIMETHOXY, but not PTERO, causes a delay in cell proliferation, particularly in mitosis progression increasing the number of cells in metaphase at the expense of prophases and ana/telophases.

Effects of resveratrol on topoisomerase II-α activity: induction of micronuclei and inhibition of chromosome segregation in CHO-K1 cells.

In recent years, a great interest has emerged in resveratrol (RSV) activity in the prevention of various pathologies including cancer. We recently showed that RSV is able to interfere with topoisomerase II-α (TOPO2) activity in cancer cells, thus inducing a delay in S-phase progression with concomitant phosphorylation of the histone H2AX. TOPO2 is mainly active in proliferating cells and is involved in the resolution of supercoiled DNA and chromosome segregation during mitosis. Here, we studied the effects of RSV in CHO-K1 cells concerning to chromosome damage and segregation as a consequence of TOPO2 inhibition. We show an increase in micronuclei and in polyploid and endoreduplicated cells due to incorrect chromosome segregation. Furthermore, since incomplete segregation can also affect the normal distribution of mitotic figures, we checked mitosis progression showing an increase in metaphase in relation to ana-telophase after RSV treatment. On the whole, our data show that RSV affects chromosome stability and segregation in proliferating cells, probably interfering with TOPO2 activity.

Resveratrol acts as a topoisomerase II poison in human glioma cells.

Recently, we demonstrated that Resveratrol (RSV), a well known natural stilbene, is able to induce a delay in S progression with a concomitant increase in γH2AX expression in U87 glioma cells. Furthermore, we showed that it inhibits the ability of recombinant human topoisomerase IIα to decatenate kDNA in vitro. Because proliferating tumor cells express topoisomerases at high levels and these enzymes are important targets of some of the most successful anticancer drugs, we tested whether RSV is able to poison topoisomerase IIα in glioma cells. Then, we monitored the increase of micronuclei in RSV treated U87 cells as a consequence of the conversion of TOPOII/DNA cleavable complexes to permanent DNA damage. Finally, we assayed the ability of RSV in modulating the expression of target proteins involved in DNA damage signalling, namely ATR, ATM, Chk1, Chk2 and γH2AX. Through a molecular modelling here we show that RSV binds at the TOPOII/DNA interface thus establishing several hydrogen bonds. Moreover, we show that RSV poisons TOPOIIα so inducing DNA damage; ATM, Chk2 and γH2AX are involved in the DNA damage signalling after RSV treatment.

Persistence of genetic damage in mice exposed to low dose of X rays.

PURPOSE: The aim of this work was to evaluate the persistence of genetic damage in CBA/J mice treated with a single irradiation of 0.1 or 1 Gy of X rays. MATERIALS AND METHODS: Peripheral blood was collected from irradiated and control mice after 30 min, 24 h, 7 days, 1, 3 and 6 months from exposure and analysed by comet assay. To investigate if the whole-body irradiation affect DNA repair, half of the sampled blood cells were in vitro-irradiated with additional 4 Gy and immediately analysed. Six months from exposure haematopoietic organs were sampled for measuring apoptotic index. RESULTS: In mice exposed to 1 Gy genetic damage was initially high and decreased during the experimental-time, while in the 0.1 Gy group damage, at first low, persisted and slightly increased. The 0.1 Gy-irradiated mice showed also a time-dependent increasing sensitivity to the in vitro-irradiation. Six months after whole-body irradiation, the percentage of apoptotic cells observed in haematopoietic compartments from 0.1 Gy-irradiated mice was significantly higher compared to controls and to 1 Gy mice. CONCLUSIONS: Results demonstrated that a single exposure to low-dose might induce long-term damage. Persistence of genetic damage might have relevant implications for estimating risk for low doses.

Ret, Abl1 (cAbl) and Trp53 gene fragmentations in comet-FISH assay act as in vivo biomarkers of radiation exposure in C57BL/6 and CBA/J mice.

The International Commission on Radiation Protection (ICRP) has lowered the dose limits for workers and for the general public exposed to ionizing radiation. Consequently, a reliable dosimetric method for monitoring possible radiation-induced damage is of great importance in radioprotection. The counting of dicentric chromosomal aberrations and of micronuclei in peripheral blood lymphocytes is unreliable when it is applied to in vivo biopsies and for low-dose exposures. Single-cell gel electrophoresis (SCGE or comet assay), although sensitive and rapid, shows high variability when applied in vivo, probably due to prompt repair of the DNA breaks and confounding environmental factors. In this paper, we describe specific in situ hybridization of Ret, Abl1 (cAbl), and Trp53 gene fragmentations on SCGE slides (comet-FISH assay) in peripheral blood cells from C57BL/6 and CBA/J mice as an indicator of radiation-induced DNA damage. The results obtained from four mice for each experimental point (0, 1, 2 and 4 Gy of X rays) discriminated in a statistically significant way the effects of all doses when fragmentations were analyzed for the Ret, Ab1 and Trp53 genes. SCGE alone, when applied to the same specimens, produced no significant results because of interindividual and experimental variability.

Resveratrol affects DNA damage induced by ionizing radiation in human lymphocytes in vitro.

Resveratrol (3,4',5-trihydroxystilbene; RSV) acts on cancer cells in several ways, inducing cell cycle delay and apoptotic death, and enhancing ionizing radiation (IR)-mediated responses. However, fewer studies have examined RSV effects on normal cells. We have treated human lymphocytes in vitro with RSV, either alone or combined with IR, to evaluate its potential use as a radioprotector. We measured the effects of RSV on induction of DNA damage, repair kinetics, and modulation of histone deacetylase activity.

Scavenging of hydroxyl radical by resveratrol and related natural stilbenes after hydrogen peroxide attack on DNA.

Resveratrol (3,5,4'-trihydroxystilbene) is of interest due to its role in prevention and therapy of degenerative diseases as cancer and aging. However, depending on its concentration and cell type studied, resveratrol activity appears conflicting. It exerts antioxidant action, as a scavenger of free radicals and as promoter of antioxidant enzyme activity, but resveratrol acts also as a pro-oxidant. Here we present experimental and theoretical studies for resveratrol and two methoxy-derivatives found in plants, pterostilbene and 3,5,4'-trimethoxystilbene. We show that both methoxy-derivatives induce less DNA damage than resveratrol. The protective effects of the three molecules against oxidative DNA damage induced by hydrogen peroxide treatment were analyzed on mammalian cells in vitro. Our data show for the first time that methoxylated derivatives of resveratrol are very efficient in reducing DNA damage: using the same concentration of the three molecules we obtain a relative reduction of 85.5% (pterostilbene), 43.7% (trimethoxystilbene) and 21.1% (resveratrol). Analysis of the crystal structures of pterostilbene and 3,5,4'-trimethoxystilbene, compared to resveratrol, show fewer intermolecular interactions and a lack of planarity, due to packing forces, which is confirmed by density functional theory (DFT) calculations. We also describe the results of DFT calculations (including water solvent effects) in which the three stilbene species scavenge the hydroxyl radical (associated with the H2O2 insult).

Cytogenetic biomonitoring on a group of petroleum refinery workers.

Workers employed in petroleum refineries are exposed to a wide range of toxic compounds (benzene, polycyclic aromatic hydrocarbons, heavy metals, etc.) with known mutagenic and carcinogenic potential. In this study, we investigated by using the cytokinesis block micronucleus (CBMN) assay on human peripheral blood lymphocytes (PBL) whether general occupational exposure in petroleum refineries resulted in early biological effects, which would be indicative of adverse health effects in the long term. In this study, out of more 500 workers enrolled in the study, 79 male subjects (46 nonsmokers and 33 smokers), employed in two different Italian petroleum refineries, and a total of 50 male control subjects (34 nonsmokers and 16 smokers) were selected by using very strict selection criteria. The comparison of chromosome damage in PBL between exposed and control populations pointed out a significant increase of micronuclei in the exposed group, correlated with the length of employment. Results confirm that smoking is the principal confounding factor for the responses. In conclusion, our results are indicative of a potential genotoxic risk related to the complex occupational exposure in petroleum refineries, despite the measures adopted in the plants, and corroborate the need to increase safety measures to avoid exposure to chemical agents.

Resveratrol induces DNA double-strand breaks through human topoisomerase II interaction.

Resveratrol, a stilbene found in grapes and wine, is one of the most interesting natural compound due to its role exerted in cancer prevention and therapy. In particular, resveratrol is able to delay cell cycle progression and to induce apoptotic death in several cell lines. Here we report that resveratrol treatment of human glioblastoma cells induces a delay in cell cycle progression during S phase associated with an increase in histone H2AX phosphorylation. Furthermore, with an in vitro assay of topoisomerase IIalpha catalytic activity we show that resveratrol is able to inhibit the ability of recombinant human TOPO IIalpha to decatenate kDNA, so that it could be considered a TOPO II poison.