TiO2 nanoparticles induce DNA double strand breaks and cell cycle arrest in human alveolar cells

TiO₂ nanoparticles (NPs) have the second highest global annual production (～3000 tons) among the metal‐containing NPs. These NPs are used as photocatalysts for bacterial disinfection, and in various other consumer products including sunscreen, food packaging, therapeutics, biosensors, surface cleaning agents, and others. Humans are exposed to these NPs during synthesis (laboratory), manufacture (industry), and use (consumer products, devices, medicines, etc.), as well as through environmental exposures (disposal). Hence, there is great concern regarding the health effects caused by exposure to NPs and, in particular, to TiO₂ NPs. In the present study, the genotoxic potential of TiO₂ NPs in A549 cells was examined, focusing on their potential to induce ROS, different types of DNA damage, and cell cycle arrest. We show that TiO₂ NPs can induce DNA damage and a corresponding increase in micronucleus frequency, as evident from the comet and cytokinesis‐block micronucleus assays. We demonstrate that DNA damage may be attributed to increased oxidative stress and ROS generation. Furthermore, genomic and proteomic analyses showed increased expression of ATM, P53, and CdC‐2 and decreased expression of ATR, H2AX, and Cyclin B1 in A549 cells, suggesting induction of DNA double strand breaks. The occurrence of double strand breaks was correlated with cell cycle arrest in G2/M phase. Overall, the results indicate the potential for genotoxicity following exposure to these TiO₂ NPs, suggesting that use should be carefully monitored. Environ. Mol. Mutagen. 56:204–217, 2015. © 2014 Wiley Periodicals, Inc.     

Identification of human papillomavirus DNA sequences in peripheral blood mononuclear cells

Polymerase chain reaction (PCR) was used to amplify and identify the presence of the DNA of human papillomavirus (HPV) types 6, 11, 16, and 18 in peripheral blood mononuclear cells (PBMCs) of women with and without urogenital HPV infections. HPV DNA of various types was found in PBMCs of 13 of 25 (52.0%) patients with urogenital HPV infections and in none of the 19 control subjects who are free of urogenital HPV infections. The presence of HPV DNA in PBMCs may impair the immunologic functions of the lymphocytes and play a role in the epidemiology of HPV infections and the pathogenesis of HPV-induced diseases.     

Ultrastructural detection of DNA strand breaks in apoptotic neural cells by in situ end-labelling techniques

Recently developed techniques based on ‘in situ end-labelling’ (ISEL) of DNA strand breaks may help to identify apoptotic cells in tissue sections. We have applied ISEL techniques at the electron microscopic (EM) level, in order to verify if ultrastructural features of apoptosis are indeed associated with evidence of DNA fragmentation, and whether cells committed to, but which have not yet entered the stage of cell death are also labelled. Terminal transferase and DNA polymerase assays were applied to thin sections of Araldite and LR Gold-embedded medulloblastomas and embryonic mouse dorsal root ganglia. Digoxigenin-labelled nucleotides were used; incorporation was demonstrated by immunogold staining. Apoptotic cells in various stages of the death process were easily labelled in both tissues. In addition, DNA fragmentation was demonstrated in cells with initial chromatin condensation, but otherwise indistinguishable from adjacent unstained cells. Our results show that EM-ISEL techniques effectively demonstrate the occurrence of DNA strand breaks in apoptotic and possibly ‘pre-apoptotic’ cells in neural tissues. Since the labelling is easily obtained on tissue that is routinely processed for electron microscopy, this technique may allow retrospective studies on archival material.     

Detection and quantitation of human papillomavirus DNA in peripheral blood mononuclear cells from blood donors

Human papillomavirus (HPV) is the etiological agent of cervical cancer. Also, HPV has been associated with anogenital cancer, oropharyngeal cancer, genital warts, and other dermatological diseases. HPV infects epithelial cells and their replication is closely linked to epithelial differentiation. The presence of HPV DNA in peripheral blood mononuclear cells (PBMC) has been reported in some patients with head and neck cancer, cervical cancer, and other genital diseases. However, the presence of HPV DNA in blood in asymptomatic subjects is still unresolved. The objective of this study was to evaluate the presence of HPV DNA in PBMC from asymptomatic blood donors. Blood samples were collected from 207 healthy Chilean blood donors. Genomic DNA was extracted from PBMC and HPV DNA detection was performed by real-time quantitative polymerase chain reaction assays with GP5+/6+ primers. HPV typing was carried out by genetic sequencing of a 140 to 150 bp fragment of the L1 gene. HPV DNA was detected in 6.8% (14/207) of blood donors. Single HPV infections were detected in seven blood donors. High-risk HPV was found in 6.3% (13/207) of cases: nine blood donors were infected with HPV-16, five with HPV-18, two with HPV-51, and one case was infected with either 32, 33, 45, 59, 66, 70, or 82. The median viral load value was 21.3 copies/mL blood or 13.4 HPV (+) cells per 10 ⁴ PBMC. These results show that HPV DNA is present in PBMC from healthy blood donors and it suggests that blood could be a new route of HPV dissemination.     

Responding to DNA double strand breaks in the nervous system

Within the nervous system appropriate responses to DNA damage are required to maintain homeostasis and prevent disease. In this tissue, DNA double-strand breaks (DSBs) initiate a molecular response to repair DNA, or in many cases, activate apoptosis. The repair of DNA DSBs occurs via nonhomologous end-joining (NHEJ) or homologous recombination (HR). These mechanistically distinct pathways are critical for maintenance of genomic integrity. During nervous system development there are discrete requirements for each DNA DSB repair pathway at different stages of development. For example, in the nervous system HR is particularly important for proliferating cells, while NHEJ is critical for differentiating cells. Inactivation of either of these pathways can lead to embryonic lethality, neurodegeneration or brain tumors. Human syndromes that result from defective responses to DNA damage often feature overt neuropathology. A prime example is the neurodegenerative syndrome ataxia telangiectasia (A-T), which results from inactivation of the ATM kinase, a crucial nexus for the cellular response to DNA DSBs. This type of DNA damage activates ATM via the Mre11-Rad50-NBS1 (MRN) complex, which leads to selective phosphorylation of ATM substrates resulting in apoptosis or cell cycle arrest and DNA repair. Furthermore, DNA DSBs resulting from chronic genotoxic stress can also result in tumorigenesis, as inactivation of either HR or NHEJ can lead to certain types of brain tumors. Thus, there are distinct requirements for each DNA DSB repair pathway during neural development, which have important implications for understanding diseases of the nervous system.     

Bioenergetic analysis of human peripheral blood mononuclear cells

Leucocytes respond rapidly to pathogenic and other insults, with responses ranging from cytokine production to migration and phagocytosis. These are bioenergetically expensive, and increased glycolytic flux provides adenosine triphosphate (ATP) rapidly to support these essential functions. However, much of this work is from animal studies. To understand more clearly the relative role of glycolysis and oxidative phosphorylation in human leucocytes, especially their utility in a translational research setting, we undertook a study of human peripheral blood mononuclear cells (MNCs) bioenergetics. Glycolysis was essential during lipopolysaccharide (LPS)-mediated interleukin (IL)−1β, IL-6 and tumour necrosis factor (TNF)-α production, as 2-deoxy-D-glucose decreased significantly the output of all three cytokines. After optimizing cell numbers and the concentrations of all activators and inhibitors, oxidative phosphorylation and glycolysis profiles of fresh and cryopreserved/resuscitated MNCs were determined to explore the utility of MNCs for determining the bioenergetics health profile in multiple clinical settings. While the LPS-induced cytokine response did not differ significantly between fresh and resuscitated cells from the same donors, cryopreservation/resuscitation significantly affected mainly some measures of oxidative phosphorylation, but also glycolysis. Bioenergetics analysis of human MNCs provides a quick, effective means to measure the bioenergetics health index of many individuals, but cryopreserved cells are not suitable for such an analysis. The translational utility of this approach was tested by comparing MNCs of pregnant and non-pregnant women to reveal increased bioenergetics health index with pregnancy but significantly reduced basal glycolysis and glycolytic capacity. More detailed analysis of discrete leucocyte populations would be required to understand the relative roles of glycolysis and oxidative phosphorylation during inflammation and other immune responses.     

Lysis of whole blood in vitro causes DNA strand breaks in human lymphocytes.

DNA damage in lymphocytes, as measured by alkaline single cell gel electrophoresis (pH 12.7), is greatly increased by the concurrent lysis of whole blood in both freshly isolated samples and in PHA-stimulated cultures over a period of 7 days. Further, there is a marked progressive increase in DNA damage with time in PHA-stimulated lymphocytes cultured in whole blood even when the lymphocytes are separated before analysis; no such increase is seen in lymphocytes cultured alone. This indicates that there are components in whole blood that can cause DNA damage in lymphocytes, with granulocytes and lysis of red blood cells likely candidates. The DNA damage is greatly reduced in granulocyte-depleted whole blood cultures, but even in these significant increases are seen at later sampling times. Consequently, careful sample preparation is of paramount importance if the Comet assay is to be successfully used to assess DNA damage in human peripheral blood lymphocytes. Further, the progressive increase in DNA damage in whole blood cultures may influence other methods using lymphocytes for population biomonitoring and may be significant for in vitro genotoxicity testing.     

Detection of human herpesvirus 8 DNA sequences in peripheral blood mononuclear cells of children

Human herpesvirus 8 (HHV-8) DNA sequences were examined in peripheral blood mononuclear cell (PBMC) DNA samples of 56 children, 15 healthy adults, and 10 renal transplant patients by the polymerase chain reaction (PCR). The PCR amplification was carried out using the published KS330₂₃₃ primer pairs to amplify HHV-8 DNA sequences. The PCR-amplified products were confirmed by Southern blot hybridization with radiolabeled 233 bp HHV-8 DNA fragment, which was cloned and sequenced from the PCR-amplified product of Kaposi's sarcoma tissue. Six PCR-amplified product of four children and two renal transplant patients were cloned and sequenced. HHV-8 DNA sequences were detected in 36 of 56 (64%) normal children, in 12 of 15 (80%) healthy adults, and in all 10 renal transplant patients by Southern blot hybridization of PCR-amplified products. Six PCR-amplified products were confirmed by sequencing. These results suggest that HHV-8 infection is prevalent in the Japanese population with infection occurring in early childhood. J. Med. Virol. 53:81–84, 1997. © 1997 Wiley-Liss, Inc.     

Study of apoptotic DNA fragmentation in human spermatozoa

The aim of our work was to define and better understand apoptosis in the spermatozoa of normal subjects, infertile patients and patients affected by specific tumoral diseases employing the method of the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling and confirming the results by electron microscopy. We studied 23 healthy, normozoospermic subjects (group A), 29 oligoasthenoteratozoospermic patients, affected by various andrological pathologies (group B), 28 patients with Hodgkin's disease (C1) and 30 patients with testicular cancer (C2). Our data demonstrate that the percentage of apoptosis in normozoospermic subjects (group A) is significantly lower than in all the other groups (B, C1, C2) (P < 0.001). This confirms that high DNA fragmentation is one of the characteristics of spermatogenetic failure. The induction of apoptosis, which can also be a basic response to neoplastic disease, can even act right up to the mature male gamete. Our results suggest that apoptosis could be the final result of various pathologies and of a deregulation of spermatogenesis control systems.     

PCR-Based assay to quantify human immunodeficiency virus type 1 DNA in peripheral blood mononuclear cells

An assay that quantifies the amount of human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood mononuclear cells has been developed. PCR amplification of the HIV-1 DNA is performed in the presence of an internal quantitation standard, and colorimetric detection of the amplified product is performed with microwell plates. The copies of HIV-1 DNA are normalized to total genomic DNA input. The assay has an analytical sensitivity of 10 input copies per amplification reaction and a three-log detection range. In an analysis of sequential samples from patients on combination therapy, HIV-1 DNA was quantifiable for all individuals tested, including those with undetectable plasma HIV-1 RNA. In a separate study, a comparison of HIV-1 DNA levels was made with a group of long-term survivors and progressors. The mean HIV-1 DNA levels were lower in the long-term survivors than in the progressors (P, 0.04). The mean HIV-1 RNA levels were also lower, but the difference was not statistically significant (P, 0.164). A quantitative DNA assay will provide an additional tool to gain insight into the natural history of infection and the continued efficacy of potent antiretroviral therapies.     

Circadian clock gene expression in human peripheral blood mononuclear cells

Circadian clock genes have been identified in humans but information regarding their expression has remained very limited. However from a basic point as well as in a diagnostic and therapeutic perspective, it is important to evaluate molecular clock gene expression. Peripheral blood mononuclear cells represent an ideal material to investigate non-invasively the human clock at the molecular level. Several studies including ours reported rhythmic expression of clock genes in these cells, with significant intersubject variability of expression. In addition, our results reveal the existence of different chronotypes of clock gene expression patterns and suggest specific regulatory mechanisms in these human cells as compared to other peripheral tissues.     

Transfection of human peripheral blood mononuclear cells using immunoporation

Immunoporation has been found to be able to efficiently transfect a wide range of human cultured cell lines. This report shows that peripheral blood mononuclear cells can also be efficiently transfected using immunoporation. The immunoporation of the cells with fluorescent TMR-Dextran, using Immunofect MG beads, indicates that transient holes of 5.4nm in diameter or larger are formed during immunoporation. The efficiencies of transfection of lymphocytes transfected with vectors coding for EGFP and lacZ were found to be within the range of 15-30% with high levels of cell viability of more than 90%. In addition, it was observed that mononuclear cells stimulated with PHA expressed transfected reporter genes with a higher efficiency. In conclusion, these results demonstrate that immunoporation using Immunofect MG beads can be used for the efficient transfection of primary lymphocytes with DNA or other macromolecules.     

Esterase staining of human peripheral blood mononuclear cells.

Recently proposed esterase staining methods for the cytochemical identification of human peripheral blood monocytes and lymphocytes in our hands gave suboptimal results. Cellular purification and recommended fixation procedures appeared to decrease the esterase reactivity of leucocyte preparations. Drying for 12-18 h without further fixation of cytocentrifuge smears was found to produce minimal loss of the staining capacity for 1-naphthyl butyrate esterase in mononuclear cells. It is hoped that the slightly modified procedure meets the need for a simple technique to define mononuclear blood cells for clinical purposes.     

Interleukin-4 gene expression in human peripheral blood mononuclear cells

Interleukin-4 (IL-4) mRNA was detected in normal human peripheral blood mononuclear cells (PBMC) stimulated with concanavalin A by Northern blot analysis. The signal was undetectable in PBMC before the stimulation, but became detectable 3 hrs after the stimulation and reached a maximum in 3-6 h and disappeared gradually thereafter. Immunosuppressive drugs such as ciclosporin, hydrocortisone and prednisolone inhibited the IL-4 mRNA expression dose dependently. Interferon-gamma did not show any inhibitory effect on IL-4 gene expression.     

Early protein synthesis in activated human peripheral blood mononuclear cells

Though B-cell division and Ig synthesis in response to pokeweed mitogen (PWM) require interaction with T-cells and monocytes, it is not clear which earlier events in B-cell activation share this requirement, and which are the result of direct interaction of mitogen with the B-cell. Having previously shown that the acceleration of lecithin synthesis in human B-cells at 16-20 hr requires both T-cells and monocytes, we now examine whether B-cells require similar interactions to increase their protein synthetic rate, another important activation event. At 21-24 hr of PWM stimulation, the stimulation index (SI) for incorporation of [35S]methionine into protein was 2.1 +/- 0.4 for unfractionated cells, 1.7 +/- 0.1 for B-cells, 2.5 +/- 0.1 for T-cells, and 3.4 +/- 0.5 for monocytes. Thus monocytes contributed substantially to early mitogen-induced protein synthesis by human peripheral blood mononuclear cells. When the monocyte/B-cell fraction (MB) and T-cell fraction (T) were mixed at various ratios in PWM-stimulated cultures, synergy was apparent at MB:T ratios of 1:1 and 1:2, indicating cell interactions augmented early mitogen-driven protein synthesis in at least one of these cell types. However, much or all of this synergy could be attributed to T-cells, whose protein synthetic response was augmented by B-cells and monocytes. In contrast, the early increase in B-cell protein synthesis appeared to be independent of cell interactions, since their SI of 1.7 was not influenced by varying the proportion of M- or T-cells over a 50-fold range. These contrasting results between two contemporary events fits the hypothesis that one (accelerated phospholipid synthesis) requires a first signal plus one or more cell interaction signals, whereas the other (accelerated protein synthesis) requires only the first signal.     

Radiation-induced DNA damage and repair in peripheral blood mononuclear cells from Nijmegen breakage syndrome patients and carriers assessed by the Comet assay

Nijmegen breakage syndrome (NBS) patients and carriers are predisposed to malignancy and are often treated with X-irradiation. In the present study, the single-cell gel electrophoresis (Comet) assay was used to examine radiation-induced DNA damage and repair in peripheral blood mononuclear cells from NBS patients (n=13) and carriers (n=36) of six unrelated families. Cells from apparently healthy donors (n=10) and from breast cancer patients with normal clinical radiosensitivity (n=10) served as controls. Cells were irradiated with 5 Gy of X-rays and assayed for initial DNA damage and for residual DNA damage after 40 min of repair; the kinetics of DNA repair also was estimated. In addition, the nuclear area of unirradiated cells was extracted from the Comet data. The initial radiation-induced DNA fragmentation indicated that cells from members of two out of six NBS families were significantly more sensitive to X-irradiation than cells from the controls. Cells from four NBS families had longer DNA repair half-time values, while cells from five NBS families had significantly increased residual DNA damage following repair. The mean nuclear area of unirradiated cells processed in the Comet assay was 1.3-fold higher in cells from all NBS families than in the controls (P<0.05). Notably, the Comet assay parameters (initial and residual DNA damage and the repair kinetics) of irradiated NBS cells predicted the carrier status of the majority (86%) of blindly tested individuals. The prediction of NBS status was higher if the nuclear area of unirradiated cells was used as the endpoint. The results of this study suggest that the impaired radiation response of NBS cells should be taken into account if radiotherapy of NBS patients and carriers is required.