Silvia Filippi

CSB protein is (a direct target of HIF-1 and) a critical mediator of the hypoxic response.

Cockayne syndrome (CS) is a rare genetic disease characterized by neurological problems, growth failure and premature ageing. Many of these features cannot simply be ascribed to the defect that CS cells display during transcription‐coupled repair. Here, we show that CSB mutant cells are unable to react to hypoxic stimuli by properly activating the hypoxia‐inducible factor‐1 (HIF‐1) pathway, a defect that is further enhanced in the event of a concomitant genotoxic stress. Furthermore, we show that CSB expression is under the control of HIF‐1 and has a critical function during hypoxic response by redistributing p300 between HIF‐1 and p53. Altogether, our data demonstrate that CSB is part of a feedback loop mechanism that modulates the biological functions of p53. The outcome of this study provides new insights into the understanding of the molecular basis of the CS phenotype and the involvement of the CSB protein in the hypoxic response pathway.

l-Carnitine enhances resistance to oxidative stress by reducing DNA damage in Ataxia telangiectasia cells

In this study, the modulating effect of L-carnitine on tert-butyl-hydroperoxide-induced DNA damage was compared with that of mannitol, a well known scavenger of hydroxyl radicals, both in normal and Ataxia telangiectasia mutated (ATM)-deficient lymphoblastoid cell lines established from A. telangiectasia (A-T) patients. The alkaline version of the comet assay was employed to measure the frequency of single-strand breaks (SSBs) and alkali-labile sites induced by t-butyl-OOH immediately after treatment and at different recovery times in normal and A-T cell lines, with and without pre-treatment with L-carnitine. In addition, both the yield of induced chromosomal damage and the effect on cell proliferation were evaluated. Our results show that pre-treatment of cells with L-carnitine produced an enhancement of the rate and extent of DNA repair in A-T cell lines at early recovery time; furthermore, in samples pre-treated with L-carnitine a reduction of all types of chromosomal aberration was observed, both in A-T and in wild-type cell lines. The reducing effect of L-carnitine pre-treatment on oxidative DNA damage was more prominent than that of pre-treatment with mannitol. In conclusion, we demonstrated a protective effect of L-carnitine on oxidative stress-induced DNA damage in A-T cells, suggesting its possible role in future pharmacological applications in A-T therapy.

CSA and CSB proteins interact with p53 and regulate its Mdm2-dependent ubiquitination

Mutations in Cockayne syndrome (CS) A and B genes (CSA and CSB) result in a rare genetic disease that affects the development and homeostasis of a wide range of tissues and organs. We previously correlated the degenerative phenotype of patients to the enhanced apoptotic response, exhibited by CS cells, which is associated with the exceptional induction of p53 protein, upon a variety of stress stimuli. Here we showed that the elevated and persistent levels of p53 displayed by CS cells are due to the insufficient ubiquitination of the p53 protein. We further demonstrated that CSA and CSB proteins associate in a unique complex with p53 and Mdm2; this interaction greatly stimulates the ubiquitination of p53 in an Mdm2-dependent manner. Tandem affinity purification and immunoprecipitations combined with mass spectrometry studies indicate that CSA and CSB associate within a Cullin Ring Ubiquitin Ligase complex responsible, under certain circumstances, for p53 ubiquitination. This study identifies CSA and CSB as the key elements of a regulatory mechanism that equilibrate beneficial and detrimental effects of p53 activity upon cellular stress. The deregulation of p53, in absence of either of the CS proteins, can potentially explain the early onset degeneration of tissues and organs observed in CS patients.

Integration of new biological and physical retrospective dosimetry methods into EU emergency response plans – joint RENEB and EURADOS inter-laboratory comparisons

Abstract Purpose: RENEB, ‘Realising the European Network of Biodosimetry and Physical Retrospective Dosimetry,’ is a network for research and emergency response mutual assistance in biodosimetry within the EU. Within this extremely active network, a number of new dosimetry methods have recently been proposed or developed. There is a requirement to test and/or validate these candidate techniques and inter-comparison exercises are a well-established method for such validation. Materials and methods: The authors present details of inter-comparisons of four such new methods: dicentric chromosome analysis including telomere and centromere staining; the gene expression assay carried out in whole blood; Raman spectroscopy on blood lymphocytes, and detection of radiation-induced thermoluminescent signals in glass screens taken from mobile phones. Results: In general the results show good agreement between the laboratories and methods within the expected levels of uncertainty, and thus demonstrate that there is a lot of potential for each of the candidate techniques. Conclusions: Further work is required before the new methods can be included within the suite of reliable dosimetry methods for use by RENEB partners and others in routine and emergency response scenarios.

Identification of Novel Proteins Co-Purifying with Cockayne Syndrome Group B (CSB) Reveals Potential Roles for CSB in RNA Metabolism and Chromatin Dynamics

The CSB protein, a member of the SWI/SNF ATP dependent chromatin remodeling family of proteins, plays a role in a sub-pathway of nucleotide excision repair (NER) known as transcription coupled repair (TCR). CSB is frequently mutated in Cockayne syndrome group B, a segmental progeroid human autosomal recessive disease characterized by growth failure and degeneration of multiple organs. Though initially classified as a DNA repair protein, recent studies have demonstrated that the loss of CSB results in pleiotropic effects. Identification of novel proteins belonging to the CSB interactome may be useful not only for predicting the molecular basis for diverse pathological symptoms of CS-B patients but also for unraveling the functions of CSB in addition to its authentic role in DNA repair. In this study, we performed tandem affinity purification (TAP) technology coupled with mass spectrometry and co-immunoprecipitation studies to identify and characterize the proteins that potentially interact with CSB-TAP. Our approach revealed 33 proteins that were not previously known to interact with CSB. These newly identified proteins indicate potential roles for CSB in RNA metabolism involving repression and activation of transcription process and in the maintenance of chromatin dynamics and integrity.

Evaluation of the effects of ellagic acid (EA) on 7,12-dimethylbenz(α) anthracene (DMBA) induced micronuclei in mammalian cells in vitro and in vivo.

We evaluated the protective effects of EA, a promising dietary constituent against degenerative diseases, on the clastogenic action of the model carcinogen DMBA in vitro on human hepatoma cells (HepG2) and in vivo on bone marrow of mice, using the frequencies of induced micronuclei as the end point. Pre-, post- and simultaneous treatments with EA and the carcinogen were carried out in vitro. Simultaneous treatment with EA caused a statistically significant increase of DMBA induced MN, suggesting a direct interaction between the two agents. No significant reduction in DMBA induced MN was found by pre- or post treatment with EA. Similar effects were observed in the toxicity assay. In in vivo experiments, EA pre-treatment did not affect the frequencies of MN in PCEs of bone marrow induced by DMBA. A good correlation was found between in vitro and in vivo experiments. Our results did not reveal any clear indication on the efficacy of EA on the induction of micronuclei by DMBA. EA by itself did not show any harmful effects.

A comparative study of the anticlastogenic effects of chlorophyllin on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 7,12-dimethylbenz (α) anthracene (DMBA) induced micronuclei in mammalian cells in vitro and in vivo.

Chlorophyllin (CHL), a water soluble derivative of chlorophyll has been shown to have both anticarcinogenic and antigenotoxic properties. We evaluated the protective effects of CHL (25μM in vitro, 4 and 100mg/kg. b.w.) on the clastogenic action of two model carcinogens, MNNG and DMBA (25μM and 2μM respectively) in vitro on human hepatoma cells (HepG2) and (40mg and 25mg/Kg/b.w. respectively) in vivo on bone marrow of mice, using the frequencies of induced micronuclei as the end point. Pre-, post- and simultaneous treatments with CHL and the carcinogen were carried out in vitro. With MNNG, only simultaneous treatment with CHL was effective in reducing the frequencies of MN, suggesting a direct interaction between CHL and MNNG. A statistically significant reduction in of DMBA induced MN was found by pre-or post treatment with CHL while a reduction (not significant) was observed by simultaneous treatment. In in vivo experiments, CHL pre-treatment did not affect the frequencies of MN in PCEs of bone marrow induced by MNNG or DMBA. However, increased the toxic effect of DMBA (reduction in percent of PCEs) was accompanied by a reduction in the induced frequencies of MN. CHL was not clastogenic in both in vitro and in vivo tests. It can be concluded that (a) CHL has a protective effect against MNNG and DMBA. This effect is dependent upon the protocol employed in in vitro experiments. In vivo, CHL did not have a protective effect against MNNG and DMBA. A protective effect of CHL against DMBA was evident only at high toxic levels.

Web based scoring is useful for validation and harmonisation of scoring criteria within RENEB.

Abstract Purpose: To establish a training data set of digital images and to investigate the scoring criteria and dose assessment of the dicentric assay within the European network of biodosimetry (RENEB), a web based scoring inter-comparison was undertaken by 17 RENEB partners. Materials and methods: Two sets of 50 high resolution images were uploaded onto the RENEB website. One set included metaphases after a moderate exposure (1.3 Gy) and the other set consisted of metaphases after a high dose exposure (3.5 Gy). The laboratories used their own calibration curves for estimating doses based on observed aberration frequencies. Results: The dose estimations and 95% confidence limits were compared to the actual doses and the corresponding z-values were satisfactory for the majority; only the dose estimations from two laboratories were too low or too high. The coefficients of variation were 17.6% for the moderate and 11.2% for the high dose. Metaphases with controversial results could be identified for training purposes. Conclusions: Overall, the web based scoring of the two galleries by the 17 laboratories produced very good results. Application of web based scoring for the dicentric assay may therefore be a relevant strategy for an operational biodosimetry assistance network.

Effect of blueberries (BB) on micronuclei induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and 7,12-dimethylbenz(a)anthracene (DMBA) in mammalian cells, assessed in in vitro and in vivo assays

The protective effect of blueberry (BB) on the clastogenic effects of MNNG and DMBA was evaluated with the induced micronucleus (MN) frequency as a biomarker, both in vitro and in vivo. Human hepatoma HepG2 cells, which contain most of the metabolic activating enzymes was used for the in vitro test. MN frequencies were determined in binucleated cells generated by blocking cytokinesis by use of cytochalasin-B. The MN frequency in vivo was determined in polychromatic erythrocytes (PCEs) from the bone marrow of treated mice. BB by itself was not toxic both in vivo and in vitro. There was no evidence of a potential physico-chemical interaction between BB and the test carcinogens in vitro. Pre-treatment with BB reduced the MN frequency induced by MNNG. But, simultaneous treatment and post-treatment with BB did not affect the frequency of MNNG-induced MN. BB did not affect the frequency of DMBA-induced MN in vitro under any test condition. Under in vivo conditions, BB reduced the frequencies of MNNG- and DMBA-induced MN in PCEs, but in the case of the protective effect of BB against DMBA a dramatic reduction in the percentage of PCEs was observed, suggesting increased cytotoxicity.

Pasta containing tartary buckwheat sprouts prevents DNA damage in spontaneously hypertensive rats

Abstract Recent studies have shown that DNA damage occurs more often in hypertensive patients than non-hypertensive individuals. Here, we analyzed the in vivo effect of pasta containing 30% of tartary buckwheat sprouts (TBSP) on spontaneously hypertensive rats (SHRs) and normotensive Wistar Kyoto rats (WKY) to elucidate if TBSP could have an anti-genotoxic effect in hypertensive animal models. Both SHRs and WKY rats were divided into two groups and fed for six weeks with 5 g of TBSP and durum wheat flour commercial pasta, respectively. Our results showed that a diet rich in TBSP has anti-genotoxic effect. Indeed, SHRs fed with TBSP exhibited a significant decrease in DNA damage (38%) and more efficient DNA repair (84%) compared to SHRs fed with commercial pasta.