Andrea Berni

The type and yield of ionising radiation induced chromosomal aberrations depend on the efficiency of different DSB repair pathways in mammalian cells.

In order to evaluate the relative role of two major DNA double strand break repair pathways, i.e., non-homologous end joining (NHEJ) and homologous recombination repair (HRR), CHO mutants deficient in these two pathways and the parental cells (AA8) were X-irradiated with various doses. The cells were harvested at different times after irradiation, representing G2, S and G1 phase at the time of irradiation, The mutant cell lines used were V33 (NHEJ deficient), Irs1SF, 51-D1 (HRR deficient). In addition to parental cell line (AA8), a revertant of V33, namely V33-155 was employed. Both types of mutant cells responded with increased frequencies of chromosomal aberrations at all recovery times in comparison to the parental and revertant cells. Mutant cells deficient in NHEJ were more sensitive in all cell stages in comparison to HRR deficient mutant cells, indicating NHEJ is the major repair pathway for DSB repair through out the cell cycle. Both chromosome and chromatid types of exchange aberrations were observed following G1 irradiation (16 and 24 h recovery). Interestingly, configurations involving both chromosome (dicentrics) and chromatid exchanges were encountered in G1 irradiated V33 cells. This may indicate that unrepaired DSBs accumulate in G1 in these mutant cells and carried over to S phase, where they are repaired by HRR or other pathways such as B-NHEJ (back up NHEJ), which appear to be highly error prone. Both NHEJ and HRR, which share some of the same proteins in their pathways, are involved in the repair of DSBs leading to chromosomal aberrations, but with a major role of NHEJ in all stages of cell cycle.

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.

Influence of glutathione levels on radiation-induced chromosomal DNA damage and repair in human peripheral lymphocytes

Endogenous thiols, especially the tripeptide-reduced glutathione (GSH), are known to play an important role in cellular defense against radiation. However, there are evidences that suggest that GSH may not be an efficient protector of DNA. The present study will determine whether modulation of endogenous GSH levels protects or potentiates the amount of chromosomal damage induced by ionizing radiation (IR). Human lymphocytes were isolated and then treated with GSH (for 1h) or buthionine sulfoximine (BSO; GSH-depleting agent for 5 h) before X-irradiation. DNA damage was analyzed by scoring chromosome aberrations (CAs) and by comet assay. The level of endogenous GSH was measured in lymphocytes treated with GSH, BSO or X-rays. A roughly 20% increase in endogenous GSH level was observed after a 3-h treatment with exogenous GSH and this reduced the frequency of all types of CA and aberrant metaphase chromosomes induced by 1 and 2 Gy of X-rays and also decreased the tail moment as determined by comet assay, suggesting radiation protection. Such uniform protection by GSH pretreatment was not visible while cells were exposed to 3 Gy or higher. Interestingly, in GSH-depleted lymphocytes, the frequency of radiation-induced CA was increased in a non-uniform manner. Therefore, an increase in the level of endogenous GSH in lymphocytes was unable to reduce chromosomal damage induced by 3 Gy or above, whereas decrease in the level of GSH enhanced the frequency of CA at all radiation doses in a non-uniform manner. It seems that GSH did not act as a radioprotector against DNA damage induced by higher dose X-rays rather it acts as a modulator of DNA repair activity.

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.

Role of chromatin structure modulation by the histone deacetylase inhibitor trichostatin A on the radio-sensitivity of ataxia telangiectasia

At present, a lot is known about biochemical aspects of double strand breaks (DBS) repair but how chromatin structure affects this process and the sensitivity of DNA to DSB induction is still an unresolved question. Ataxia telangiectasia (A-T) patients are characterised by very high sensitivity to DSB-inducing agents such as ionising radiation. This radiosensitivity is revealed with an enhancement of chromosomal instability as a consequence of defective DNA repair for a small fraction of breaks located in the heterochromatin, where they are less accessible. Besides, recently it has been reported that Ataxia Telangiectasia Mutated (ATM) mediated signalling modifies chromatin structure. In order to study the impact of chromatin compaction on the chromosomal instability of A-T cells, the response to trichostatin-A, an histone deacetylase inhibitor, in normal and A-T lymphoblastoid cell lines was investigated testing its effect on chromosomal aberrations, cell cycle progression, DNA damage and repair after exposure to X-rays. The results suggest that the response to both trichostatin-A pre- and continuous treatments is independent of the presence of either functional or mutated ATM protein, as the reduction of chromosomal damage was found also in the wild-type cell line. The presence of trichostatin-A before exposure to X-rays could give rise to prompt DNA repair functioning on chromatin structure already in an open conformation. Differently, trichostatin-A post-treatment causing hyperacetylation of histone tails and reducing the heterochromatic DNA content might diminish the requirement for ATM and favour DSBs repair reducing chromosomal damage only in A-T cells. This fact could suggest that trichostatin-A post-treatment is favouring the slow component of DSB repair pathway, the one impaired in absence of a functionally ATM protein. Data obtained suggest a fundamental role of chromatin compaction on chromosomal instability in A-T cells.

Exposure to polycyclic aromatic hydrocarbons in women from Poland, Serbia and Italy – relation between PAH metabolite excretion, DNA damage, diet and genotype (the EU DIEPHY project)

Abstract Exposure of the general population to polycyclic aromatic hydrocarbons (PAH) is ubiquitous. The aim of this study was to analyze biomarkers associated with the uptake of PAH in 428 non-smoking women from Łodz (Poland), Viterbo (Italy), Belgrade (Serbia) and from the Pančevo area, where the petrochemical complex was destroyed by the air raids in 1999. Urinary excretion of PAH metabolites was lowest in Italian women, intermediary for Serbian and highest in Polish women, who predominantly excreted hydroxy phenanthrenes as metabolites of phenanthrene. Bulky DNA adduct levels were highest in Italian and Polish women. Genotype or PAH ambient air levels could not explain the dissimilarities between the study groups with respect to biomarker patterns, which probably reflected differences in life style-associated factors.

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.

Study on X-ray-induced apoptosis and chromosomal damage in G2 human lymphocytes in the presence of pifithrin-α, an inhibitor of p53

The aim of this study is to investigate the role of the cell-cycle phase in cells exposed to radiation and chemicals in relation to the cellular response. The analysis was focused on the G2 cell-cycle phase, exploring the impact of p53 inhibition in human lymphocytes irradiated with X-rays in the presence or absence of pifithrin-α (PFT-α), a p53-specific inhibitor. Lymphocytes, 44h after stimulation to proliferate, were X-irradiated with 0.5Gy both in the presence or the absence of PFT-α and post-treated with a pulse of 5-bromodeoxyuridine (BrdUrd) to distinguish cells in the S- or G2-phase at the moment of irradiation. At early sampling times after X-ray exposure the following parameters were analysed: cellular proliferation, apoptosis, chromosomal aberrations and p53 expression. The results show an enhancement of apoptotic cells in G2 at early sampling times after irradiation and no differences in terms of chromosomal aberration induction both in cells treated with X-rays alone and in cells treated with X-rays plus PFT-α. Expression of p53 was not detectable at all recovery times. The results suggest a p53-independent apoptotic pathway acting at early times after X-ray exposure in G2 lymphocytes. Furthermore, the same yield of X-ray-induced chromatid breaks was observed both in the presence or absence of PFT-α implying that in G2 X-irradiated lymphocytes this inhibitor of the p53 protein does not affect DNA repair.

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.