Maria Giuseppa Grollino

Sperm DNA fragmentation induced by DNAse I and hydrogen peroxide: an in vitro comparative study among different mammalian species.

Sperm DNA damage may have adverse effects on reproductive outcome. Sperm DNA breaks can be detected by several tests, which evaluate DNA integrity from different and complementary perspectives and offer a new class of biomarkers of the male reproductive function and of its possible impairment after environmental exposure. The remodeling of sperm chromatin produces an extremely condensed nuclear structure protecting the nuclear genome from adverse environments. This nuclear remodeling is species specific, and differences in chromatin structure may lead to a dissimilar DNA susceptibility to mutagens among species. In this study, the capacity of the comet assay in its two variants (alkaline and neutral) to detect DNA/chromatin integrity has been evaluated in human, mouse, and bull sperm. The hypothesis that chromatin packaging might influence the amount of induced and detectable DNA damage was tested by treating sperm in vitro with DNAse I, whose activity is strictly dependent upon its DNA accessibility. Furthermore, hydrogen peroxide (H2O2) was used to assess whether spermatozoa of the three species showed a different sensitivity to oxidative stress. DNAse I-induced damage was also assessed by the sperm chromatin structure assay and the TUNEL assay, and the performances of these two assays were compared and correlated with the comet assay results. Results showed a different sensitivity to DNAse I treatment among the species with human sperm resulting the most susceptible. On the contrary, no major differences among species were observed after H2O2 treatment. Furthermore, the three tests show a good correlation in revealing sperm with DNA strand breaks.

Biodistribution and acute toxicity of a nanofluid containing manganese iron oxide nanoparticles produced by a mechanochemical process

Superparamagnetic iron oxide nanoparticles are candidate contrast agents for magnetic resonance imaging and targeted drug delivery. Biodistribution and toxicity assessment are critical for the development of nanoparticle-based drugs, because of nanoparticle-enhanced biological reactivity. Here, we investigated the uptake, in vivo biodistribution, and in vitro and in vivo potential toxicity of manganese ferrite (MnFe2O4) nanoparticles, synthesized by an original high-yield, low-cost mechanochemical process. Cultures of murine Balb/3T3 fibroblasts were exposed for 24, 48, or 72 hours to increasing ferrofluid concentrations. Nanoparticle cellular uptake was assessed by flow-cytometry scatter-light measurements and microscopy imaging after Prussian blue staining; cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony-forming assays. After a single intravenous injection, in vivo nanoparticle biodistribution and clearance were evaluated in mice by Mn spectrophotometric determination and Prussian blue staining in the liver, kidneys, spleen, and brain at different posttreatment times up to 21 days. The same organs were analyzed for any possible histopathological change. The in vitro study demonstrated dose-dependent nanoparticle uptake and statistically significant cytotoxic effects from a concentration of 50 μg/mL for the MTT assay and 20 μg/mL for the colony-forming assay. Significant increases in Mn concentrations were detected in all analyzed organs, peaking at 6 hours after injection and then gradually declining. Clearance appeared complete at 7 days in the kidneys, spleen, and brain, whereas in the liver Mn levels remained statistically higher than in vehicle-treated mice up to 3 weeks postinjection. No evidence of irreversible histopathological damage to any of the tested organs was observed. A comparison of the lowest in vitro toxic concentration with the intravenously injected dose and the administered dose of other ferrofluid drugs currently in clinical practice suggests that there might be sufficient safety margins for further development of our formulation.

Kinetics of γ-H2AX induction and removal in bone marrow and testicular cells of mice after X-ray irradiation

Male germ cells have been shown to differ in their DNA damage response (DDR) with respect to somatic cells. In addition, DDR pathways are modulated along spermatogenesis, accompanying profound chromatin modifications. Histone H2AX phosphorylation is a fundamental step of DDR. Few data are available on the long-term kinetics of phosphorylated H2AX (γ-H2AX) after in vivo irradiation. We have investigated, by microscopic and flow cytometric immunochemistry, γ-H2AX induction and removal in testicular cells of irradiated mice, in comparison with bone marrow cells. In unirradiated testicular cells, much higher levels of γ-H2AX were measured by flow cytometry with respect to bone marrow cells. Irradiation induced a redistribution of γ-H2AX into discrete foci detectable by microscopy. In irradiated bone marrow, the percentage of labelled cells peaked at 1 h and rapidly declined, in agreement with data on in vitro cell lines. In contrast, spermatocytes and round spermatids showed persistent labelling until 48 h. During this time, in spermatids, topological changes were observed in γ-H2AX foci from a pattern of many uncountable dots to a pattern of few large spots. Observations of testicular sections confirmed this trend in the reduction of foci number in spite of substantially invariable percentages of labelled cells in the analysed timeframe. To assess whether γ-H2AX persistence in testicular cells was due to unrepaired DNA breaks, we performed comet assay and immunofluorescence analysis of Mdc1, a marker of DDR different from γ-H2AX. Comet assay showed that most breaks were repaired within 2 h. Forty-eight hours after irradiation, contrary to γ-H2AX foci that remained detectable in 80% of initially labelled cells, Mdc1 foci were observed in only 20-30% of cells. These data suggest that, at long times after irradiation, mechanisms additional to impairment of DNA break repair may account for the long persistence of γ-H2AX foci in male germ cells.

The usefulness of combining traditional sperm assessments with in vitro heterospermic insemination to identify bulls of low fertility as estimated in vivo

To date, no single in vitro assessment can estimate bull fertility. This research was aimed at evaluating the ability of a series of laboratory assessments to assign 50 Holstein Friesian bulls grouped as low (ER-NRR<-1.5), medium (-0.5<ER-NRR<+0.5) and high (ER-NRR≥+1.5) fertility based on estimated relative non-return rates (ER-NRR), to the two categories of low and medium-high fertility. Heterospermic insemination with a Piedmontese reference bull was employed to define an index of competitive binding ability (CBI) to the zona pellucida using fluorochrome-labeled sperm, and a competitive fertility index (CFI) using embryo paternal assignment by single nucleotide polymorphism (SNP). Furthermore, kinetic parameters, membrane integrity and sperm DNA/chromatin integrity (% DFI) were assayed. Low fertility bulls had lesser (P<0.05) values for total motility and membrane integrity, and a greater value of % DFI as compared to medium and high fertility groups. A modest (P<0.001) correlation was reported among ER-NRR and total motility (r=0.30), progressive motility (r=0.26), membrane integrity (r=0.43) and % DFI (r=-0.26). While % DFI alone allowed the identification of 70% lowly fertile bulls, combining membrane integrity, average path velocity and CBI allowed for identification of 78% of the lowly fertile sires. Paternal assignment by SNPs resulted in 96% of successful assignments and could provide an alternative support to microsatellites for in vivo studies based on heterospermic fertilization for estimating fertility.

Direct and Delayed X-Ray-Induced DNA Damage in Male Mouse Germ Cells

Sperm DNA integrity is essential for the accurate transmission of paternal genetic information. Various stages of spermatogenesis are characterized by large differences in radiosensitivity. Differentiating spermatogonia are susceptible to radiation‐induced cell killing, but some of them can repair DNA damage and progress through differentiation. In this study, we applied the neutral comet assay, immunodetection of phosphorylated H2AX (γ‐H2AX) and the Sperm Chromatin Structure Assay (SCSA) to detect DNA strand breaks in testicular cells and spermatozoa at different times following in vivo X‐ray irradiation. Radiation produced DNA strand breaks in testicular cells that were repaired within the first few hours after exposure. Spermatozoa were resistant to the induction of DNA damage, but non‐targeted DNA lesions were detected in spermatozoa derived from surviving irradiated spermatogonia. These lesions formed while round spermatids started to elongate within the testicular seminiferous tubules. The transcription of pro‐apoptotic genes at this time was also enhanced, suggesting that an apoptotic‐like process was involved in DNA break production. Our results suggest that proliferating spermatogonia retain a memory of the radiation insult that is recognized at a later developmental stage and activates a process leading to DNA fragmentation. Environ. Mol. Mutagen. 2012.

Radioresistance in a tumour cell line correlates with radiation inducible Ku 70/80 end-binding activity.

Purpose: The aims of the present study were to better understand the role of Ku 80, which is involved in double-strand break repair in mammalian cells in the mechanism of radiation resistance and to verify the possibility of increasing cell radiosensitivity by targeted inhibition of Ku autoantigen 80 (Ku 80). Materials and methods: Western blot and electrophoretic mobility shift assay (EMSA) were performed on the human bladder carcinoma cell line RT112 (radioresistant) and on the human colorectal carcinoma cell line SW48 (radiosensitive) to assess the expression levels of DNA-dependent protein kinase (DNA-PK) components and the DNA-binding activity of the Ku 70/80 heterodimer after exposure to radiation, respectively. Ku 80 silencing was carried out with the use of small interfering RNA (siRNA). Results: Greater differences in the DNA-binding activity of Ku 70/80 and Ku 80 phosphorylation level were observed in RT112 as compared to SW48 after X-ray treatment. There is no correlation between Ku expression and DNA-binding activity at lower doses. A significant increase in nuclear Ku 80 expression was observed one hour after the exposure, only at the higher doses, while the DNA-PK catalytic subunits (DNA-PKcs) and Ku 70 levels did not change significantly. Inhibition of Ku 80 expression by siRNA induced radiosensitivity in the RT112 cell line. Conclusions: Our data demonstrate that in a bladder tumour cell line up-regulation of Ku end-binding activity without any marked change in Ku expression underlie radiation resistance.

Bladder Transitional Cell Carcinomas: A Comparative Study of Washing and Tumor Bioptic Samples by DNA Flow Cytometry and FISH Analyses

Objective and Methods: We compared information obtained from samples of tumor biopsy and bladder washing to evaluate the representatives of the latter type of sampling. Both types of samples from 44 cases of superficial bladder TCCs and one papilloma were analyzed by FCM, to evaluate cellular DNA content, and FISH, to detect numerical aberration of chromosome 9. Results: The use of both tumor and washing samples by FCM and FISH analyses evidenced alterations in 95% of cases. FCM evidenced higher aneuploid frequency in bladder washing than in bioptic specimens. In bladder washing, FISH analysis showed higher frequency of monosomy and lower frequency of trisomy than in biopsy. No correlation was found between histological grade and centromeric chromosome 9 loss while correlation was evidenced with centromeric 9 gain. Conclusion: Irrigation specimens, analyzed by FCM and FISH, can complement information obtained by biopsy in cytodiagnosis and follow–up of patients with bladder TCC.

Chromosome 9 aberrations by fluorescence in situ hybridisation in bladder transitional cell carcinoma

To investigate the role of the monosomy 9 in bladder carcinogenesis, 96 cases of superficial bladder transitional cell carcinoma (TCC) were studied and followed periodically for around 3 years (mean+/-standard error of the mean (SEM); 3.46+/-0.34 years). Samples from bladder washings were analysed by fluorescent in situ hybridisation (FISH) to detect numerical anomalies of chromosome 9. Moreover, to evaluate the relative under representation of this chromosome, we detected numerical changes of chromosome 8 and DNA ploidy by flow cytometric analysis (FCM). Chromosome 8 copy number were related to FCM DNA ploidy and both were related with tumour grade. Monosomy 9 did not correlate with tumour grade, stage, chromosome 8 aneuploidies and abnormal DNA content, but correlated with tumour progression. Comparing the results in the primary and subsequent tumours, we observed an increase in the frequency of aneuploidies by FCM, associated with an increase of chromosome 8 polysomies. The mean chromosome 9 copy number/nucleus remained nearly the same in most of the primary and invasive tumours. Our results confirm that monosomy 9 is an early event and that it is retained during tumour progression and invasion and that the loss occurs before the tetraploidisation process. The relationship between the presence of a sub-population with monosomy 9 and tumour progression suggests the presence of a region that could have a role in the progression of superficial bladder TCC.