Lucia Celotti

The influence of cytochrome P450 1A1 and glutathione S-transferase M1 genotypes on biomarker levels in coke-oven workers

Abstract The present study has the aim of evaluating gene-environment interaction on the levels of different biomarkers in coke-oven workers exposed to PAH. In order to assess whether the levels of some biomarkers (PAH-DNA adducts, nitro-PAH adducts to Hb and MN frequency) could be modulated by the genetic metabolic polymorphisms for CYP1A1 and GSTM1, we analysed in 76 coke-oven workers and 18 controls the CYP1A1 (MspI and Ile/Val sites) and the GSTM1 genotypes by a PCR assay. In individuals with shared set-up of CYP1A1 or GSTM1 genotypes, we analysed how the specified biomarkers correlated with total PAH exposure (urinary levels of 1-hydroxypyrene) both by a stratified analysis and logistic regression modelling. Statistically significant (P = 0.03 and P = 0.01) higher percentages of the more susceptible GSTM1− subjects compared to the GSTM1+ subjects and of the more susceptible CYP1A1 Ile/Val individuals compared to the CYP1A1 Ile/Ile individuals were detected for high levels of PAH-DNA adducts in the high exposure group (namely high levels of 1-OHP). A statistically significant association was observed between increased PAH-DNA adduct levels and the more susceptible GSTM1- genotype (P.O.R. = 4.18, P = 0.03) in a logistic regression modelling and a significant interaction between PAH exposure and GSTM1-genotype was found for PAH-DNA adducts. No effect of these metabolic genotypes was observed for MN frequency and nitro-PAH adducts to Hb. In conclusion, a gene-environment interaction between PAH exposure and two metabolic genotypes involved in activation (CYP1A1) and detoxification (GSTM1) of PAHs, respectively, has been identified.

Influence of GSTM1, GSTT1, GSTP1, and EPHX gene polymorphisms on DNA adduct level and HPRT mutant frequency in coke-oven workers

To evaluate the influence of individual susceptibility factors on the level of polyaromatic (PAH) hydrocarbon DNA adducts and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutants in peripheral lymphocytes, 70 coke-oven workers exposed to PAH were genotyped for four metabolic enzyme polymorphisms of potential importance in PAH metabolism. The examined genetic polymorphisms concerned glutathione S-transferases M1 (GSTM1; gene deletion; 96 workers), T1 (GSTT1; gene deletion), P1 (GSTP1; Ile-->Val substitution at codon 104 or Ile-->Val at codon 104 and Val-->Ala at codon 113), and microsomal epoxide hydrolase (EPHX; Tyr-->His substitution at codon 113 and His-->Arg at codon 139). The workers were classified in a high- and low-exposure group on the basis of urinary concentration of 1-pyrenol. The GSTM1 null genotype increased the number of DNA adducts in smoking coke-oven workers with high PAH exposure. DNA adducts were affected by PAH-exposure in non-smokers and in GSTM1 null smokers and by smoking in GSTM1 null individuals. In a multiple linear regression analysis, the interaction of the GSTM1 genotype was statistically significant (p = 0.04) with smoking (yes/no) and of borderline significance (p = 0.06) with PAH-exposure (high/low). As smoking also increased urinary 1-pyrenol, the genotype modification seemed to concern DNA adducts due to smoking rather than occupational exposure. GSTT1 positive individuals showed an elevated level of DNA adducts in comparison with GSTT1 null subjects (p = 0.04), and EPHX genotypes associated with slow hydroxylation reaction yielded a higher (p = 0.05) HPRT mutant frequency than fast EPHX genotypes; these findings were, however, based on small numbers of subjects and need to be clarified in further studies. In conclusion, our findings indicate that homozygous deletion of GSTM1 results in an increased sensitivity to genotoxic PAHs in tobacco smoke, which is seen as an increase in aromatic DNA adducts in blood mononuclear cells.

Analysis of miRNA and mRNA Expression Profiles Highlights Alterations in Ionizing Radiation Response of Human Lymphocytes under Modeled Microgravity

Background Ionizing radiation (IR) can be extremely harmful for human cells since an improper DNA-damage response (DDR) to IR can contribute to carcinogenesis initiation. Perturbations in DDR pathway can originate from alteration in the functionality of the microRNA-mediated gene regulation, being microRNAs (miRNAs) small noncoding RNA that act as post-transcriptional regulators of gene expression. In this study we gained insight into the role of miRNAs in the regulation of DDR to IR under microgravity, a condition of weightlessness experienced by astronauts during space missions, which could have a synergistic action on cells, increasing the risk of radiation exposure. Methodology/Principal Findings We analyzed miRNA expression profile of human peripheral blood lymphocytes (PBL) incubated for 4 and 24 h in normal gravity (1 g) and in modeled microgravity (MMG) during the repair time after irradiation with 0.2 and 2Gy of γ-rays. Our results show that MMG alters miRNA expression signature of irradiated PBL by decreasing the number of radio-responsive miRNAs. Moreover, let-7i*, miR-7, miR-7-1*, miR-27a, miR-144, miR-200a, miR-598, miR-650 are deregulated by the combined action of radiation and MMG. Integrated analyses of miRNA and mRNA expression profiles, carried out on PBL of the same donors, identified significant miRNA-mRNA anti-correlations of DDR pathway. Gene Ontology analysis reports that the biological category of “Response to DNA damage” is enriched when PBL are incubated in 1 g but not in MMG. Moreover, some anti-correlated genes of p53-pathway show a different expression level between 1 g and MMG. Functional validation assays using luciferase reporter constructs confirmed miRNA-mRNA interactions derived from target prediction analyses. Conclusions/Significance On the whole, by integrating the transcriptome and microRNome, we provide evidence that modeled microgravity can affects the DNA-damage response to IR in human PBL.

The cellular uptake of meta-tetra (hydroxyphenyl)chlorin entrapped in organically modified silica nanoparticles is mediated by serum proteins

Nanosized objects made of various materials are gaining increasing attention as promising vehicles for the delivery of therapeutic and diagnostic agents for cancer. Photodynamic therapy (PDT) appears to offer a very attractive opportunity to implement drug delivery systems since no release of the sensitizer is needed to obtain the therapeutic effect and the design of the nanovehicle should be much easier. The aim of our study was to investigate the use of organic-modified silica nanoparticles (NPs) for the delivery of the second-generation photosensitizer meta-tetra(hydroxyphenyl)chlorin (mTHPC) to cancer cells in vitro. mTHPC was entrapped in NPs (approximately 33 nm diameter) in a monomeric form which produced singlet oxygen with a high efficiency. In aqueous media with high salt concentrations, the NPs underwent aggregation and precipitation but their stability could be preserved in the presence of foetal bovine serum. The cellular uptake, localization and phototoxic activity of mTHPC was determined comparatively in human oesophageal cancer cells after its delivery by the NPs and the standard solvent ethanol/poly(ethylene glycol) 400/water (20:30:50, by vol). The NP formulation reduced the cellular uptake of mTHPC by about 50% in comparison to standard solvent while it did not affect the concentration-dependent photokilling activity of mTHPC and its intracellular localization. Fluorescence resonance energy transfer measurements, using NPs with mTHPC physically entrapped and a cyanine covalently linked, and ultracentrifugation experiments indicated that mTHPC is transferred from NPs to serum proteins when present in the medium. However, the coating of the NP surface with poly(ethylene glycol) largely prevented the transfer to proteins. In conclusion, mTHPC is rapidly transferred from the uncoated nanoparticles to the serum proteins and then internalized by the cells as a protein complex, irrespective of its modality of delivery.

In vitro and in vivo characterization of temoporfin-loaded PEGylated PLGA nanoparticles for use in photodynamic therapy

AIMS In this study we evaluated temoporfin-loaded polyethylene glycol (PEG) Poly-(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) as a new formulation for potential use in cancer treatment. MATERIALS & METHODS NPs were characterized for their photophysical properties, temoporfin release, cellular uptake and intracellular localization, and dark and photocytotoxicities of temoporfin by using A549, MCF10A neoT and U937 cell lines. In vivo imaging was performed on athymic nude-Foxn1 mice. RESULTS Temoporfin was highly aggregated within the NPs and the release of temoporfin monomers was faster from PEGylated PLGA NPs than from non-PEGylated ones. PEGylation significantly reduced the cellular uptake of NPs by the differentiated promonocytic U937 cells, revealing the stealth properties of the delivery system. Dark cytotoxicity of temoporfin delivered by NPs was less than that of free temoporfin in standard solution (Foscan(®), Biolitec AG [Jena, Germany]), whereas phototoxicity was not reduced. Temoporfin delivered to mice by PEGylated PLGA NPs exhibits therapeutically favorable tissue distribution. CONCLUSION These encouraging results show promise in using PEGylated PLGA NPs for improving the delivery of photosensitizers for photodynamic therapy.

DNA repair in modeled microgravity: Double strand break rejoining activity in human lymphocytes irradiated with γ-rays

Cell response to ionising radiation depends, besides on genetic and physiological features of the biological systems, on environmental conditions occurring during DNA repair. Many data showed that microgravity, experienced by astronauts during space flights or modeled on Earth, causes apoptosis, cytoskeletal alteration, cell growth inhibition, increased frequency of mutations and chromosome aberrations. In this study, we analysed the progression of the rejoining of double strand breaks (DSBs) in human peripheral blood lymphocytes (PBLs) irradiated with gamma-rays and incubated in static condition (1 g) or in modeled microgravity (MMG). gamma-H2AX foci formation and disappearance, monitored during the repair incubation, showed that the kinetics of DSBs rejoining was different in the two gravity conditions. The fraction of foci-positive cells decreased slower in MMG than in 1 g at 6 and 24 h after irradiation (P<0.01) and the mean number of gamma-H2AX foci per nucleus was significantly higher in MMG than in 1g at the same time-points (P<0.001). In the same samples we determined apoptotic level and the rate of DSB rejoining during post-irradiation incubation. A significant induction of apoptosis was observed in MMG at 24 h after irradiation (P<0.001), whereas at shorter times the level of apoptosis was slightly higher in MMG respect to 1 g. In accordance with the kinetics of gamma-H2AX foci, the slower rejoining of radiation-induced DSBs in MMG was observed by DNA fragmentation analyses during the repair incubation; the data of pulsed-field gel electrophoresis assay showed that the fraction of DNA released in the gel was significantly higher in PBL incubated in MMG after irradiation with respect to cells maintained in 1 g. Our results provide evidences that MMG incubation during DNA repair delayed the rate of radiation-induced DSB rejoining, and increased, as a consequence, the genotoxic effects of ionising radiation.

The efficiency of homologous recombination and non-homologous end joining systems in repairing double-strand breaks during cell cycle progression.

This study investigated the efficiency of Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair systems in rejoining DNA double-strand breaks (DSB) induced in CCD-34Lu cells by different γ-ray doses. The kinetics of DNA repair was assessed by analyzing the fluorescence decrease of γ-H2AX foci measured by SOID (Sum Of Integrated Density) parameter and counting foci number in the time-interval 0.5–24 hours after irradiation. Comparison of the two methods showed that the SOID parameter was useful in determining the amount and the persistence of DNA damage signal after exposure to high or low doses of ionizing radiation. The efficiency of DSB rejoining during the cell cycle was assessed by distinguishing G1, S, and G2 phase cells on the basis of nuclear fluorescence of the CENP-F protein. Six hours after irradiation, γ-H2AX foci resolution was higher in G2 compared to G1 cells in which both NHEJ and HR can cooperate. The rejoining of γ-H2AX foci in G2 phase cells was, moreover, decreased by RI-1, the chemical inhibitor of HR, demonstrating that homologous recombination is at work early after irradiation. The relevance of HR in DSB repair was assessed in DNA-PK-deficient M059J cells and in CCD-34Lu treated with the DNA-PKcs inhibitor, NU7026. In both conditions, the kinetics of γ-H2AX demonstrated that DSBs repair was markedly affected when NHEJ was absent or impaired, even in G2 phase cells in which HR should be at work. The recruitment of RAD51 at DSB sites was, moreover, delayed in M059J and in NU7026 treated-CCD-34Lu, with respect to DNA-PKcs proficient cells and continued for 24 hours despite the decrease in DNA repair. The impairment of NHEJ affected the efficiency of the HR system and significantly decreased cell survival after ionizing radiation, confirming that DSB rejoining is strictly dependent on the integrity of the NHEJ repair system.

Modeled Microgravity Affects Cell Response to Ionizing Radiation and Increases Genomic Damage

Abstract Canova, S., Fiorasi. F., Mognato. M., Grifalconi, M, Reddi, E., Russo, A. and Celotti, L. “Modeled Microgravity” Affects Cell Response to Ionizing Radiation and Increases Genomic Damage. Radiat. Res. 163, 191–199 (2005). The aim of this work was to assess whether “modeled microgravity” affects cell response to ionizing radiation, increasing the risk associated with radiation exposure. Lymphoblastoid TK6 cells were irradiated with various doses of γ rays and incubated for 24 h in a modeled microgravity environment obtained by the Rotating Wall Vessel bioreactor. Cell survival, induction of apoptosis and cell cycle alteration were compared in cells irradiated and then incubated in 1g or modeled microgravity conditions. Modulation of genomic damage induced by ionizing radiation was evaluated on the basis of HPRT mutant frequency and the micronucleus assay. A significant reduction in apoptotic cells was observed in cells incubated in modeled microgravity after γ irradiation compared with cells maintained in 1g. Moreover, in irradiated cells, fewer G2-phase cells were found in modeled microgravity than in 1g, whereas more G1-phase cells were observed in modeled microgravity than in 1g. Genomic damage induced by ionizing radiation, i.e. frequency of HPRT mutants and micronucleated cells, increased more in cultures incubated in modeled microgravity than in 1g. Our results indicate that modeled microgravity incubation after irradiation affects cell response to ionizing radiation, reducing the level of radiation-induced apoptosis. As a consequence, modeled microgravity increases the frequency of damaged cells that survive after irradiation.

The DNA-Damage Response to γ-Radiation Is Affected by miR-27a in A549 Cells

Perturbations during the cell DNA-Damage Response (DDR) can originate from alteration in the functionality of the microRNA-mediated gene regulation, being microRNAs (miRNAs), small non-coding RNAs that act as post-transcriptional regulators of gene expression. The oncogenic miR-27a is over-expressed in several tumors and, in the present study, we investigated its interaction with ATM, the gene coding for the main kinase of DDR pathway. Experimental validation to confirm miR-27a as a direct regulator of ATM was performed by site-direct mutagenesis of the luciferase reporter vector containing the 3′UTR of ATM gene, and by miRNA oligonucleotide mimics. We then explored the functional miR-27a/ATM interaction under biological conditions, i.e., during the response of A549 cells to ionizing radiation (IR) exposure. To evaluate if miR-27a over-expression affects IR-induced DDR activation in A549 cells we determined cell survival, cell cycle progression and DNA double-strand break (DSB) repair. Our results show that up-regulation of miR-27a promotes cell proliferation of non-irradiated and irradiated cells. Moreover, increased expression of endogenous mature miR-27a in A549 cells affects DBS rejoining kinetics early after irradiation.

Meta-tetra(hydroxyphenyl)chlorin-loaded liposomes sterically stabilised with poly(ethylene glycol) of different length and density: characterisation, in vitro cellular uptake and phototoxicity.

We studied the effects of density and thickness of PEG coating on in vitro cellular uptake, and dark- and photo-toxicity of liposomal formulations (Fospeg) of the photodynamic agent meta-tetrahydroxyphenyl chlorin (m-THPC). The cellular uptake of various Fospeg formulations was determined by flow cytometry in CCD-34Lu human normal fibroblasts and A549 lung cancer cells. Dark and light-induced cytotoxicity was measured by MTS assay after exposure to increasing concentrations of Fospeg only and followed by irradiation with red light. Intracellular localization of m-THPC delivered by Fospeg was determined by fluorescence microscopy. The studies were carried out in comparison with m-THPC delivered by the standard solvent. In the dark all Fospeg formulations were less cytotoxic than m-THPC in standard solvent (ethanol/poly(ethylene glycol 400/water; 20 : 30 : 50 by vol.) and cytotoxicity decreased by increasing PEGylation. m-THPC delivered as Fospeg was internalised by endocytosis and localised mainly in the Golgi apparatus and endoplasmic reticulum. The efficiency of cellular uptake of Fospeg was reduced by 30-40% with respect to m-THPC in standard solution causing a slight reduction of the phototoxicity but without serious impairment of the efficacy of the treatment. Our study suggests that PEGylated liposomes are promising nanocarriers for the delivery of photosensitisers for photodynamic therapy because they reduce dark cytotoxicity while preserving therapeutic efficacy.