DIPARTIMENTO DI BIOLOGIA
prof.ssa Renata Cozzi site
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|Prof. Renata Cozzi - Associated Professor of Genetics|
|Dr Tommaso Cornetta - Ph. D. Student|
|Silvia Sterpone - M. Sc. Student|
- DNA REPAIR -
Mammalian cells are constantly exposed to ubiquitous environmental and endogenous genotoxic agents. The biologic consequences of mutations and persisting lesions range from the onset of carcinogenesis, genetic disorders, and apoptosis to general malfunctioning of cells that contribute to aging. Distinct mechanisms have evolved to repair different types of DNA damage and to maintain genomic integrity. Investigators have demonstrated that persons showing severely compromised repair capacity have increased mutation rates, genomic instability, and an increased risk of cancer . In general, healthy persons also differ in intrinsic capacity in repairing DNA damage, and this variation may result from alterations in gene expression or could be a result of the polymorphisms of genes involved in different
repair pathways. The repair mechanisms that have particular relevance are those that remove minor changes in helical structures, like base excision repair (BER) and nucleotide excision repair (NER), or those that participate in DNA double-strand breaks resolution, like homology-directed repair (HDR) and non-homologous end-joining (NHEJ) repair. A suitable tool to study DNA repair capacity in human lymphocytes is the alkaline Comet assay. This test, also known as single cell gel electrophoresis (SCGE), is a rapid,
reliable, and sensitive method of detecting direct DNA damage at the single cell level, with respect to single-strand breaks, DNA interstrand cross-links, and base damages that appear as endonuclease-sensitive sites. It has been widely used to measure both in vitro and in vivo DNA damage and repair after the exposure of mammalian cells to various genotoxic agents such as chemicals and ionizing and nonionizing radiation. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining at successive time intervals (cellular repair assay). The aim of this project is to study the initial DNA damage induced by X-ray treatment and the associated subsequent DNA temporal repair patterns in human peripheral blood cells of healthy subjects, in relation to some DNA repair genotypes.
- RADIOSENSITIVITY -
Many efforts have been dedicated to the possibility of developing biomarkers of individual sensitivity to ionizing radiation. Enhanced sensitivities and variability in processing the induced DNA damage can be responsible of both higher risk of developing cancer and elevated normal tissue radiosensitivity. It has been hypothesized that these complex traits are influenced by genetic factors, in particular by “minor genetic determinants”, depending on the interplay of several gene products. In view of the importance of DNA repair in cell and tissue response to radiation, genetic variants in genes responsible for DNA damage repair pathways are suitable candidates in search for the genetic basis of radiosensitivity. The purpose of this project is to examine the association of polymorphisms in genes involved in DNA damage repair with cellular radiosensitivity expressed by DNA primary damage and chromatid breaks. Among the cytogenetic test systems on peripheral blood lymphocytes, the G2 assay can be considered as the standard measure of chromosomal radiosensitivity both in normal and cancer patients.
- CYSTIC FIBROSIS -
Cystic fibrosis (CF), one of the most common single-gene lethal disease in Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductant regulator (CFTR) gene, which result in its altered expression and functions. Although it has been well demonstrated that CFTR is a chloride (Cl-) channel located in the apical membrane of most secretory cells and that the altered protein causes dense mucous epithelial secretions, additional functions have been proposed to explain the multiple phenotypic abnormalities associated with the disease. In fact, the clinical course of the disease varies widely, involving the gastrointestinal and pulmonary apparatus, liver, pancreas and also reproductive tract. Some of these clinical variations between CF patients is genetic, but conferred by genes other than CFTR. CF disease is also characterized by a combined increase of reactive oxygen species production and reduced antioxidant protection. The loss of function of CFTR channel results in altered permeability to GSH that contributes to the imbalance in the oxidant/antioxidant system. CF patients also present a constitutive increased intracellular free radical generation, due to altered electron transport activity in the mitochondria. The project consist in analysing by the Comet assay the presence of DNA damage in leucocytes from CF patients, matched with healthy controls. Furthermore we are going to genotypize all the subjects for genes involved in DNA damage repair and antioxidant defences. In particular we will assess the presence of SNPs in XRCC1 and OGG1 genes involved in Base Excision Repair and, because the Glutathione S-transferases (GST’s) play an important role in the protection against oxidative stress, the presence of genetic variants in three members of GST superfamily, GSTM1, GSTT1 and GSTP1.
- RESVERATROL -
A wide variety of polyphenolic compounds [flavonoids (anthocyanins, catechins) and non-flavonoids (stilbenes)], found mainly in vegetables and fruits, especially in grapes and their derivatives, have attracted attention for their beneficial effects in protecting human health. One of these compounds, resveratrol (3,4’,5, trihydroxystilbene), which is present in significant amounts both in the usual Mediterranean consumption (red wine, grapes, peanuts, green vegetables and other edible spermatophytes) and in many oriental herbal beverages (green tea) and medicines, has recently been the focus of a lot of attention. Population studies have suggested that the intake of moderate levels of red wine, decreases mortality associated with coronary artery diseases probably because resveratrol, one of its constituents, inhibits polymorphonuclear leukocyte function and platelet aggregation and prevents superoxidedependent inflammatory responses induced by ischemia. Since the early 1990s, increasing studies in the field of oncology have been devoted to the assessment of the anticarcinogenic and cancer chemopreventive activities of resveratrol. Many studies suggested that it is capable of regulating the three stages of carcinogenesis (initiation, promotion and progression). However, in spite of the wide variety of studies, the molecular mechanisms underlying its antitumorigenic activities are still not defined. In general, the analyses were focused on two possible mechanisms of action: arrest/delay of cell replication and induction of apoptosis. Cell cycle modulation by resveratrol has been observed in normal and tumour cell lines as well as the induction of apoptosis as a
consequence of its antiproliferative activity. We analyzed different biological effects of Resveratrol in vitro:
- we studied the antioxidant activity actually exerted by the agent against reactive oxygen species induced by H2O2 treatments in CHO cells. Our attention has been focused on two major potential mechanisms:
scavenging activity and interference with oxidative metabolism, by the analysis of three important targets: intracellular oxidation (Dichlorofluorescein Test), primary DNA damage (Comet Assay) and fixed DNA damage (chromosomal aberrations). Cells were treated with a single H2O2 dose (2×10−4 M) in order to induce Reactive Oxygen Species and than challenged with Resveratrol to test its ability in modulating damage. Two experimental protocols have been applied: (i) simultaneous treatment and (ii) a 3 h Resveratrol pre-treatment. In our experimental conditions Resveratrol does not appear able, ‘per se’, to induce primary DNA damage whereas a slight increase in endogenous oxidation and chromosomal aberrations at the highest dose have to be noticed. In combined treatments the molecule appears to differently affect primary and fixed DNA damage.
- we investigated the cellular response to treatments with X rays and resveratrol, alone or in combination, in terms of DNA damage, cell cycle delays and induction of apoptosis. Lymphoblastoid cells AHH-1 were treated and analysed at successive sampling times in order to study the induction of DNA breaks using the Comet assay and the induction of apoptosis and cell cycle modulation
through cytofluorimetric analysis.
- we have looked into whether resveratrol is able to modulate cell cycle progression in human glioblastoma cells and whether this modulation could be associated with a regulation of GJs expression in cancer cells. With this aim in mind we have performed a cytofluorimetric multiparameter assay to quantify the presence of GJs in U87 glioma cells treated or not with resveratrol and/or X rays.
- PARKINSON DISEASE -
Levodopa (L-DOPA), the metabolic precursor of dopamine, is widely used as a pharmacological agent for the symptomatic treatment of Parkinson’s disease. However, the effectiveness of L-DOPA therapy declines on continuous use, this being accompanied by dyskinesia and on-off phenomena. Another potential problem with the use of L-DOPA in treatment of Parkinson's disease arises from the fact that L- DOPA metabolism or autoxidation can give rise to radical species, hydrogen peroxide (H2O2), semiquinones, and quinones. The quinones generated are thought to mediate toxicity by covalent binding to nucleophilic groups of biological macromolecules. The H2O2 resulting from the metabolism or autoxidation of L-DOPA and dopamine can be easily reduced in the presence of ferrous iron (Fe2+) through the Fenton reaction originating the hydroxyl radical (HO*), which is considered the most damaging free radical for living cells. This may be of particular importance in Parkinson's disease, due to the fact that iron is increased in the substantia nigra in Parkinson's disease. Recently, it has been postulated that during the preclinical phase of Parkinson's disease the turnover rate of dopamine may increase to compensate the loss of dopamine containing neurones. This increase in the rate of dopamine utilization is likely to accelerate the neurodegenerative process through the generation of quinones, semi- quinones and H2O2, resulting from the oxidative metabolism of dopamine.
In this project we are measuring DNA strand breaks by using Alkalyne Comet Assay, before and after L-DOPA treatment, in peripheral blood leucocytes of PD patients. Peripheral blood mononuclear cells (PBLs) from LD-treated patients represent suitable models for the study of the consequence of LD therapy on the redox homeostasis in ex-vivo dopaminergic cells. Indeed, PBLs synthesize endogenous DA through a tyrosine-hydroxylase (TH)-dependent pathway
and express vesicular monoamine transporter (VMAT), dopamine transporters (DAT) and both D1 and D2-type dopamine receptors (DA-R). The expression ofLAT1 on their surface enables them to mimic the uptake of LD in SNpc neurons. Several findings confirmed that PBLs from untreated and LD-treated PD patients may undergo oxidative and apoptotic damage.
- BIOMONITORING STUDIES -
Several biomarkers are available to assess genetic risk or cancer risk in human populations. They are commonly classified into three groups:
An exposure biomarker should be able to indicate both the presence and the amount of an environmental agent, but they aren’t able to determine whether an environmental agent causes a toxicological damage.
An effect biomarker should be able to identify and to quantify the biological effect of an environmental exposure.
A genetic susceptibility biomarker is the measure of an inherited or acquired characteristic that could modulate organism responses to environmental exposure (Xenobiotics metabolism gene polymorphisms & DNA repair gene polymorphisms).
Genetic biomonitoring of human populations exposed to potential mutagens/carcinogens is an early warning system for genetic disease or cancer.
Human biomonitoring can be performed using different cytogenetic markers: Chromosome aberrations (CAs), Micronuclei (MN), Sister Chromatid Exchanges (SCEs) and COMET ASSAY.
CAs, MN and SCEs are the most extensively used markers of early biological effects of DNA damaging.
On the other hand the Comet Assay is a rapid, reliable and visual method for assessing DNA damage quantitatively in single cells and is established as a valuable tool in fundamental DNA damage and repair studies in population biomonitoring.
We performed different biomonitoring studies:
A multi-biomarker analysis of DNA damage in automobile painters (see reference 2 below);
Molecular biomonitoring of a population of nurses handling antineoplastic drugs;
Influence of glutathione s-transferase polymorphisms on the genotoxic effect induced by tobacco smoke
1: Cornetta T, Festa F, Testa A, Cozzi R. DNA damage repair and genetic polymorphisms: assessment of individual sensitivity and repair capacity.Int J Radiat Oncol Biol Phys. 2006 Oct 1;66(2):537-45.
2: Testa A, Festa F, Ranaldi R, Giachelia M, Tirindelli D, De Marco A, Owczarek M, Guidotti M, Cozzi R. A multi-biomarker analysis of DNA damage in automobile painters. Environ Mol Mutagen. 2005 Oct;46(3):182-8.
3: Fiore M, Festa F, Cornetta T, Ricordy R, Cozzi R. Resveratrol affects X-ray induced apoptosis and cell cycle delay in human cells in vitro. Int J Mol Med. 2005 Jun;15(6):1005-12.
4: Festa F, Cristaldi M, Ieradi LA, Moreno S, Cozzi R. The Comet assay for the detection of DNA damage in Mus spretus from Donana National Park. Environ Res. 2003 Jan;91(1):54-61.
5: Sanchez-Lamar A, Fuentes JL, Fonseca G, Capiro N, Ferrer M, Alonzo A, Baluja L, Cozzi R, De Salvia R, Fiore M, Llagostera M. Assessment of the potential genotoxic risk of Phyllantus orbicularis HBK aqueous extract using in vitro and in vivo assays. Toxicol Lett. 2002 Dec 15;136(2):87-96.
6: De Salvia R, Festa F, Ricordy R, Perticone P, Cozzi R. Resveratrol affects in a different way primary versus fixed DNA damage induced by H(2)O(2) in mammalian cells in vitro. Toxicol Lett. 2002 Sep 5;135(1-2):1-9.
7: Festa F, Aglitti T, Duranti G, Ricordy R, Perticone P, Cozzi R. Strong antioxidant activity of ellagic acid in mammalian cells in vitro revealed by the comet assay. Anticancer Res. 2001 Nov-Dec;21(6A):3903-8.
8: De Marco A, De Salvia R, Polani S, Ricordy R, Sorrenti F, Perticone P, Cozzi R, D'Ambrosio C, De Simone C, Guidotti M, Albanesi T, Duranti G, Festa F, Gensabella G, Owczarek M. Evaluation of genotoxic and cytotoxic properties of pesticides employed in Italian agricultural practices. Environ Res. 2000 Jul;83(3):311-21.
WebMaster Tommaso Cornetta