Sabrina Canova

Detection of micronuclei in gill cells and haemocytes of mussels exposed to benzo[a]pyrene

Mediterranean mussels were exposed to benzo[a]pyrene for 2 days at doses which had previously caused the formation of specific adducts in gill DNA. Micronuclei and other nuclear abnormalities were detected in gill cells and haemocytes in order to ascertain the induction of cytogenetic damage in two different target cells in parallel. A number of procedural details were examined initially to improve the quality of slides obtained from mussel cells. Adequate cytological preparations were obtained when gill cells and haemocytes were suspended, respectively, in Alsever and sea water with EDTA, cytospun and fixed with absolute methanol. In the exposed mussels, micronuclei significantly increased in both the large gill cells (the main cell type) and the agranular haemocytes. Granular haemocytes, cells present in variable proportions between individual mussels, did not show cytogenetic damage except at the highest B[a]P doses. In the same slides, steady levels of binucleated cells were detected, whereas the incidence of other nuclear abnormalities was significantly higher in the exposed compared with control mussels. Precise knowledge of the replication kinetics of gill cells and haemocytes is still lacking.

Tissue dose, DNA adducts, oxidative DNA damage and CYP1A-immunopositive proteins in mussels exposed to waterborne benzo[a]pyrene

A collaborative study was performed on Mediterranean mussels (Mytilus galloprovincialis) exposed to a wide dose-range (0.5-1000 ppb) of benzo[a]pyrene (B[a]P). We selected this model polycyclic aromatic hydrocarbon in order to confirm the formation of a specific DNA adduct, previously detected in gill DNA, and to clarify the in vivo effects of this mutagenic chemical requiring host-metabolism in mussels. B[a]P concentration reached consistently higher values in the digestive gland than in other analyzed tissues of mussels exposed to B[a]P for 2 or 3 days. With the exception of some values at 1000 ppb of B[a]P. DNA adduct levels increased significantly with the dose in gills and digestive gland and ranged from 0.054 to 0.789 adducts per 10(8) nucleotides (mean values per dose-point). Conversely, more complex dose-response relationships were found by detecting in parallel the levels of an oxidative DNA lesion (8-OHdG) and of CYP1A-immunopositive proteins (the latter measured in the digestive gland only). Overall, the formation of DNA adducts, the evidence of oxidative DNA damage, and changes in CYP1A-immunopositive protein levels support the hypothesis that B[a]P can induce DNA damage in mussels through a number of different molecular mechanisms.

Formation of DNA adducts in the gill tissue of Mytilus galloprovincialis treated with benzo[a]pyrene.

Abstract The formation of DNA adducts was evident after treatment of spawned or resting Mediterranean mussels (Mytilus galloprovincialis Lmk.) with 0.5–100 μg/l of benzo[a]pyrene for 2 and 3 days. Reference DNA samples, in vitro radiolabelled with 0.5, 5, 50μM 3H-anti (±)-B[a]P-diol-epoxide, were initially used to compare two DNA purification procedures. Following a standard four-step extraction starting with phenol, in comparison to a simplified single-step extraction without phenol, we obtained lower yields of bound radioactivity in the reference DNA samples. After simplified DNA purification and nuclease PI enhanced 32P-postlabelling assay we detected a reproducible dose-dependent increase of a specific spot in gills of mussels treated with B[a]P, although this spot was present in low amounts. Short (2 days) and prolonged (27 days) pretreatment of mussels with a polychlorinated biphenyl mixture, Aroclor 1254, did not increase the levels of B[a]P-related adducts. On the whole, these results indicate the formation of detectable amounts of DNA reactive intermediates in gills of mussels treated with B[a]P. Although the pathway of formation and the molecular identity of the specific adducts remain unclear, their presence suggests that polycyclic aromatic hydrocarbons may cause genetic damage in marine mussels.

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.

DNA Adducts in Mytilus Galloprovincialis and Zosterisessor Ophiocephalus Collected from PAC-Polluted and Reference Sites of the Venice Lagoon

Abstract Mediterranean mussels (Mytilus galloprovincialis) and bottom-dwelling grass gobies (Zosterisessor ophiocephalus) were collected from the Venice lagoon both in front of the open sea and from the industrial area of Porto Marghera. DNA adducts were measured by means of the nuclease P1-enhanced 32P-DNA postlabelling assay. Autoradiograms of gill DNA from animals of the industrial area showed a tract of unresolved adducts along the diagonal radioactive zone. Such adducts were detected at low but significant levels in different sampling periods only in mussels and fish collected at Porto Marghera. The presence of bulky aromatic DNA adducts in native mussels is consistent with previous results on the appearance of a weak adduct in gill DNA of Mytilus galloprovincialis treated with benzo[a]pyrene. However, the mechanisms leading to DNA damage and the fate of the initial lesions in marine invertebrates exposed to genotoxic agents are still poorly understood.

Interaction of activated Cdc42-associated tyrosine kinase ACK2 with HSP90

ACK2 (activated Cdc42-associated tyrosine kinase 2) is a specific downstream effector for Cdc42, a member of the Rho family of small G-proteins. ACK2 interacts with clathrin, an endocytic vesicle coating protein, and SH3PX1, a sorting nexin, and is involved in clathrin-mediated endocytosis. While searching for proteins that interact with ACK2, we found that HSP90 (heat-shock protein 90) binds to ACK2. Analysis of a series of truncation mutants of ACK2 has defined the regions within the kinase domain of ACK2 that are required for binding to HSP90. The binding of HSP90 to ACK2 is blocked upon treatment with geldanamycin, an HSP90-specific ATPase inhibitor, and is required for the in vivo kinase activity of ACK2 and its association with Cdc42. Overall, our data suggest a novel mechanism of regulation in which HSP90 serves as a regulatory component in an ACK2 functional complex and plays a role in sustaining its kinase activity.

Genetic Damage Induced by In Vitro Irradiation of Human G0 Lymphocytes with Low-Energy Protons (28 keV/μm): HPRT Mutations and Chromosome Aberrations

Abstract Mognato, M., Bortoletto, E., Ferraro, P., Baggio, L., Cherubini, R., Canova, S., Russo, A. and Celotti, L. Genetic Damage Induced by In Vitro Irradiation of Human G0 Lymphocytes with Low-Energy Protons (28 keV/μm): HPRT Mutations and Chromosome Aberrations. Radiat. Res. 160, 52–60 (2003). Cell survival, mutations and chromosomal effects were studied in primary human lymphocytes exposed in G0 phase to a proton beam with an incident energy of 0.88 MeV (incident LET of 28 keV/μm) in the dose range 0.125–2 Gy. The curves for survival and mutations at the hypoxanthine-guanine phosphoribosyl transferase locus were obtained by fitting the experimental data to linear and linear-quadratic equations, respectively. In the dose interval 0–1.5 Gy, the α parameters of the curves were 0.42/Gy and 3.6 × 10−6 mutants/Gy, respectively. The mutation types at the HPRT locus were analyzed by multiplex-PCR in 94 irradiated and 41 nonirradiated clones derived from T lymphocytes from five healthy donors. All clones showed a normal multiplex-PCR pattern and were classified as point mutations. Chromosome aberration data were fitted as a linear function of dose (α = 0.62 aberrations per cell Gy−1). By irradiating G0 lymphocytes from a single subject with 28 keV/μm protons and γ rays, an RBE of 6.07 was obtained for chromosome aberrations. An overinvolvement of chromosome 9 relative to chromosome 7 was found in chromosome breaks after chromosome painting analysis.

The DNA-Damage Response to Ionizing Radiation in Human Lymphocytes

The human genome is constantly subjected to DNA damage derived from endogenous and exogenous sources. Normal cellular metabolism can give raise to DNA damage through free radicals production and replication errors, whereas environmental agents, such as ultraviolet (UV) and ionizing radiation (IR), induce specific types of lesions. DNA damage can ultimately lead to genomic instability and carcinogenesis if not properly addressed, thus an elaborate network of proteins has evolved in cells to maintain genome integrity through a pathway termed the DNA-damage response (DDR). DDR allows DNA damage detection, signal propagation and transduction to a multitude of effector proteins, which promote cell survival and activate cell cycle arrest to allow DNA repair. When cells are unable to properly repair DNA, apoptosis or senescence pathways may be triggered, thus eliminating the possibility of passing on damaged or unrepaired genetic material to its progeny. The ultimate goal of DDR is to protect the integrity of genetic information and its faithful transmission, either to DNA by replication or to mRNA by transcription. Therefore, dysregulation of DDR pathway can contribute to carcinogenesis and developmental defects. Ionizing radiation represents a mutagen agent to which human population is exposed due to environmental, professional or accidental reasons. The biological effects of IR depend on the quality and the dose of radiation and on the cell type. Linear energy transfer (LET) represents the energy lost per unit distance as an ionizing particle travels through a material, and it is used to quantify the effects of IR on biological specimens. High-LET radiation (i.e. alpha-particles, neutrons, protons) are densely IR since they lose the energy throughout a small distance, causing dense ionization along their track with high localized multiple DNA damage. Low-LET radiation, such as X and -rays, are sparsely IR since they produce ionizations sparsely along their track and, hence, almost homogeneously within a cell. The biological effect of high-LET radiations are in general much higher than those of low-LET radiations with the same energy. This is because high-LET radiation deposits most of its energy within the volume of one cell and the damage to DNA is therefore larger (Anderson et al., 2002; Brenner & Ward, 1992; Prise et al., 2001). Radiation is potentially harmful to humans, because the ionization it produces can significantly alter the structure of molecules within a living cell.The exposure to ionizing radiation elicits a complex cell response to overcome the dangerous effects of DNA-radiation interaction, such as reactive oxygen species (ROS) production, base oxidation and DNA breaks formation (i.e. single-