Co-assessment of cell cycle and micronucleus frequencies demonstrates the influence of serum on the in vitro genotoxic response to amorphous monodisperse silica nanoparticles of varying sizes.

Serum proteins have been shown to modulate the cytotoxic and genotoxic responses to nanomaterials. The aim was to investigate the influence of serum on the induction of micronuclei (MN) by nanoparticles (NPs) of different sizes. Therefore, A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles (SNPs) were used as models. Assessment of the cell viability, cell cycle changes and induction of MN by SNPs ranging from 12 to 174 nm was performed in presence or absence of serum, applying the in vitro flow cytometry-based MN assay. Here, it has been demonstrated that serum has an influence on these end points, with a lower cell viability in absence of serum compared with the presence of serum. Further, cell cycle changes, specifically, G1 and S-phase arrest, were observed in absence of serum for four out of six SNPs tested. A size-dependent MN induction was observed: larger SNPs being more active in absence of serum. In addition, the serum influence was characterised by a size-dependency for cytotoxic and genotoxic effects, with a higher influence of serum for smaller particles. The data indicate that the in vitro micronucleus assay in presence and absence of serum could be advised for hazard assessment because it demonstrates a higher sensitivity in serum-free conditions than in conditions with serum. However, this recommendation applies only if the cell line used is able to proliferate under serum-free conditions because cell division is a prerequisite for MN expression.

Smoking patterns, depression, and sociodemographic variables among Flemish women during pregnancy and the postpartum period.

BACKGROUND: Relationships among feelings of depression, smoking behavior, and educational level during pregnancy have been documented. Feelings of depression may contribute to persistent smoking during pregnancy. No longitudinal studies assessing feelings of depression in women with different antepartum and postpartum smoking patterns are available. OBJECTIVES: The aim was to determine relationships between depressive symptoms, sociodemographic characteristics, and smoking pattern during and after pregnancy. METHODS: An observational, prospective, noninterventional study was conducted. Data were collected during two stages of pregnancy (T0: <;16 weeks and T1: 32-34 weeks) and postpartum (T2: >6 weeks) in 523 Flemish women. Feelings of depression (measured using the Beck Depression Inventory [BDI]), smoking behavior, and sociodemographic variables were analyzed using a general linear mixed model implemented in SAS Proc MIXED. RESULTS: Smokers and initial smokers reported significantly more depressive symptoms at all time points compared with recent ex-smokers, nonsmokers, and initial nonsmokers (p <; .001). The three-way interaction among time point, smoking pattern, and educational level was significant (p = .02). Evolution of mean BDI over time differed by educational level. Among participants with a secondary school certificate or less, differences were observed between smokers and nonsmokers, recent ex-smokers and initial nonsmokers, and nonsmokers and initial nonsmokers. Among participants with a college or university degree, no differences were observed. DISCUSSION: A wide variety of smoking patterns were observed during pregnancy and early postpartum. Smoking patterns were associated with depression and showed complex interactions with educational level. Assessment and intervention for both smoking and depression are needed throughout the perinatal period to support the health of mothers, their infants, and families.

Cytokine production by co-cultures exposed to monodisperse amorphous silica nanoparticles: the role of size and surface area.

The aim of this study was to test the influence of nanoparticle size and surface area (SA) on cytokine secretion by co-cultures of pulmonary epithelial cells (A549), macrophages (differentiated THP-1 cells) and endothelium cells (EA.hy926) in a two-compartment system. We used monodisperse amorphous silica nanoparticles (2, 16, 60 and 104 nm) at concentrations of 5 µg/cm² cell culture SA or 10 cm² particle SA/cm². A549 and THP-1 cells were exposed to nanoparticles for 24h, in the presence of EA.hy926 cells cultured in an insert introduced above the bi-culture after 12h. Supernatants from both compartments were recovered and TNF-α, IL-6, IL-8 and MIP-1α were measured. Significant secretion of all cytokines was observed for the 2 nm particles at both concentrations and in both compartments. Larger particles of 60 nm induced significant cytokine secretion at the dose of 10 cm² particle SA/cm². The use of multiple cellular types showed that cytokine secretion in single cell cultures is amplified or mitigated in co-cultures. The release of pro-inflammatory mediators by endothelial cells not directly exposed to nanoparticles indicates a possible endothelium activation after inhalation of silica particles. This work shows the role of size and SA in cellular response to amorphous nanosilica.

Oxidative stress induced by pure and iron-doped amorphous silica nanoparticles in subtoxic conditions.

Amorphous silica nanoparticles (SiO2-NPs) have found broad applications in industry and are currently intensively studied for potential uses in medical and biomedical fields. Several studies have reported cytotoxic and inflammatory responses induced by SiO2-NPs in different cell types. The present study was designed to examine the association of oxidative stress markers with SiO2-NP induced cytotoxicity in human endothelial cells. We used pure monodisperse amorphous silica nanoparticles of two sizes (16 and 60 nm; S16 and S60) and a positive control, iron-doped nanosilica (16 nm; SFe), to study the generation of hydroxyl radicals (HO·) in cellular-free conditions and oxidative stress in cellular systems. We investigated whether SiO2-NPs could influence intracellular reduced glutathione (GSH) and oxidized glutathione (GSSG) levels, increase lipid peroxidation (malondialdehyde (MDA) and 4-hydroxyalkenal (HAE) concentrations), and up-regulate heme oxygenase-1 (HO-1) mRNA expression in the studied cells. None of the particles, except SFe, produced ROS in cell-free systems. We found significant modifications for all parameters in cells treated with SFe nanoparticles. At cytotoxic doses of S16 (40-50 µg/mL), we detected weak alterations of intracellular glutathione (4 h) and a marked induction of HO-1 mRNA (6 h). Cytotoxic doses of S60 elicited similar responses. Preincubation of cells being exposed to SiO2-NPs with an antioxidant (5 mM N-acetylcysteine, NAC) significantly reduced the cytotoxic activity of S16 and SFe (when exposed up to 25 and 50 µg/mL, respectively) but did not protect cells treated with S60. Preincubation with NAC significantly reduced HO-1 mRNA expression in cells treated with SFe but did not have any effect on HO-1 mRNA level in cell exposed to S16 and S60. Our study demonstrates that the chemical composition of the silica nanoparticles is a dominant factor in inducing oxidative stress.

Influence of serum on in situ proliferation and genotoxicity in A549 human lung cells exposed to nanomaterials.

In this work in situ proliferation of A549 human lung epithelial carcinoma cells exposed to nanomaterials (NMs) was investigated in the presence or absence of 10% serum. NMs were selected based on chemical composition, size, charge and shape (Lys-SiO(2), TiO(2), ZnO, and multi walled carbon nanotubes, MWCNTs). Cells were treated with NMs and 4h later, cytochalasin-B was added. 36 h later, cell morphology was analyzed under a light microscope. Nuclearity was scored to determine the cytokinesis-block proliferation index (CBPI). CBPI, based on percentage of mono-, bi- and multi-nucleated cells, reflects cell toxicity and cell cycle delay. For some conditions depending on NM type (TiO(2) and MWCNT) and serum concentration (0%) scoring of CBPI was impossible due to overload of agglomerated NMs. Moreover, where heavy agglomeration occurs, micronuclei (MN) detection and scoring under microscope was prevented. A statistically significant decrease of CBPI was found for ZnO NM suspended in medium in the absence or presence of 10% serum at 25 µg/ml and 50 µg/ml, respectively and for Lys-SiO(2) NM at 3.5 µg/ml in 0% serum. Increase in MN frequency was observed in cells treated in 10% serum with 50 µg/ml ZnO. In 0% serum, the concentrations tested led to high toxicity. No genotoxic effects were induced by Lys-SiO(2) both in the absence or presence of serum up to 5 µg/ml. No toxicity was detected for TiO(2) and MWCNTs in both 10% and 0% serum, up to the dose of 250 µg/ml. Restoration of CBPI comparable to untreated control was shown for cells cultured without serum and treated with 5 µg/ml of Lys-SiO(2) NM pre-incubated in 100% serum. This observation confirms the protective effect of serum on Lys-SiO(2) NM cell toxicity. In conclusion in situ CBPI is proposed as a simple preliminary assay to assess both NMs induced cell toxicity and feasibility of MN scoring under microscope.

The cytotoxic activity of amorphous silica nanoparticles is mainly influenced by surface area and not by aggregation.

The aggregation state of NP has been a significant source of difficulty for assessing their toxic activity and great efforts have been done to reduce aggregation of and/or to disperse NP in experimental systems. The exact impact of aggregation on toxicity has, however, not been adequately assessed. Here we compared in vitro the cytotoxic activity of stable monodisperse and aggregated silicon-based nanoparticles (SNP) without introducing a dispersing agent that may affect NP properties. SNP aggregates (180 nm) were produced by controlled electrostatic aggregation through addition of KCl to a Ludox SM sol (25 nm) followed by stabilization and extensive dialysis. The size of the preparations was characterized by TEM and DLS; specific surface area and porosity were derived from N(2) sorption measurements. Macrophage (J774) and fibroblast (3T3) cell lines were exposed to monodisperse or aggregate-enriched suspensions of SNP in DMEM in absence of serum. The cytotoxic activity of the different preparations was assessed by the WST1 assay after 24h of exposure. Parameters that determined the cytotoxic activity were traced by comparing the doses of the different preparations that induced half a maximal reduction in WST1 activity (ED(50)) in both cell lines. We found that ED(50) (6-9 µg/ml and 15-22 µg/ml, in J774 and 3T3, respectively) were hardly affected upon aggregation, which was consistent with the fact that the specific surface area of the SNP, a significant determinant of their cytotoxic activity, was unaffected upon aggregation (283-331 m(2)/g). Thus studying small aggregated NP could be as relevant as studying disperse primary NP, when aggregates keep the characteristics of NP, i.e. a high specific surface area and a nanosize dimension. This conclusion does, however, not necessarily hold true for other toxicity endpoints for which the determinants may be different and possibly modified by the aggregation process.

Methodological approaches influencing cellular uptake and cyto-(geno) toxic effects of nanoparticles.

Methods are needed to assess cytotoxicity and genotoxicity of nanoparticles (NPs). The influence of serum and the use of cytochalasin-B were assessed on the cellular uptake of amorphous silica NPs (SNPs) and their biological effects. Our observations indicate that some methodological approaches may modulate the outcome of the assay. Therefore the experimental design and choice of the assays are of great importance in nanotoxicology.

Antiretroviral therapy CNS penetration and HIV-1-associated CNS disease.

OBJECTIVE: The impact of different antiretroviral agents on the risk of developing or surviving CNS disease remains unknown. The aim of this study was to investigate whether using antiretroviral regimens with higher CNS penetration effectiveness (CPE) scores was associated with reduced incidence of CNS disease and improved survival in the UK Collaborative HIV Cohort (CHIC) Study. METHODS: Adults without previous CNS disease, who commenced combination antiretroviral therapy (cART) between 1996 and 2008, were included (n = 22,356). Initial and most recent cART CPE scores were calculated. CNS diseases were HIV encephalopathy (HIVe), progressive multifocal leukoencephalopathy (PML), cerebral toxoplasmosis (TOXO), and cryptococcal meningitis (CRYPTO). Incidence rates and overall survival were stratified by CPE score. A multivariable Poisson regression model was used to identify independent associations. RESULTS: The median (interquartile range) CPE score for initial cART regimen increased from 7 (5-8) in 1996-1997 to 9 (8-10) in 2000-2001 and subsequently declined to 6 (7-8) in 2006-2008. Differences in gender, HIV acquisition risk group, and ethnicity existed between CPE score strata. A total of 251 subjects were diagnosed with a CNS disease (HIVe 80; TOXO 59; CRYPTO 56; PML 54). CNS diseases occurred more frequently in subjects prescribed regimens with CPE scores ≤ 4, and less frequently in those with scores ≥ 10; however, these differences were nonsignificant. Initial and most recent cART CPE scores ≤ 4 were independently associated with increased risk of death. CONCLUSION: Clinical status at time of commencing cART influences antiretroviral selection and CPE score. This information should be considered when utilizing CPE scores for retrospective analyses.

The nanosilica hazard: another variable entity.

Silica nanoparticles (SNPs) are produced on an industrial scale and are an addition to a growing number of commercial products. SNPs also have great potential for a variety of diagnostic and therapeutic applications in medicine. Contrary to the well-studied crystalline micron-sized silica, relatively little information exists on the toxicity of its amorphous and nano-size forms. Because nanoparticles possess novel properties, kinetics and unusual bioactivity, their potential biological effects may differ greatly from those of micron-size bulk materials. In this review, we summarize the physico-chemical properties of the different nano-sized silica materials that can affect their interaction with biological systems, with a specific emphasis on inhalation exposure. We discuss recent in vitro and in vivo investigations into the toxicity of nanosilica, both crystalline and amorphous. Most of the in vitro studies of SNPs report results of cellular uptake, size- and dose-dependent cytotoxicity, increased reactive oxygen species levels and pro-inflammatory stimulation. Evidence from a limited number of in vivo studies demonstrates largely reversible lung inflammation, granuloma formation and focal emphysema, with no progressive lung fibrosis. Clearly, more research with standardized materials is needed to enable comparison of experimental data for the different forms of nanosilicas and to establish which physico-chemical properties are responsible for the observed toxicity of SNPs.

Exploring the aneugenic and clastogenic potential in the nanosize range: A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles as models.

We explored how to assess the genotoxic potential of nanosize particles with a well validated assay, the in vitro cytochalasin-B micronucleus assay, detecting both clastogens and aneugens. Monodisperse Stöber amorphous silica nanoparticles (SNPs) of three different sizes (16, 60 and 104 nm) and A549 lung carcinoma cells were selected as models. Cellular uptake of silica was monitored by ICP-MS. At non-cytotoxic doses the smallest particles showed a slightly higher fold induction of micronuclei (MNBN). When considering the three SNPs together, particle number and total surface area appeared to account for MNBN induction as they both correlated significantly with the amplitude of the effect. Using nominal or cellular dose did not show statistically significant differences. Likewise, alkaline comet assay and FISH-centromeric probing of MNBN indicated a weak and not statistically significant induction of oxidative DNA damage, chromosome breakage and chromosome loss. This line of investigation will contribute to adequately design and interpret nanogenotoxicity assays.

Influence of size, surface area and microporosity on the in vitro cytotoxic activity of amorphous silica nanoparticles in different cell types.

Identifying the physico-chemical characteristics of nanoparticles (NPs) that drive their toxic activity is the key to conducting hazard assessment and guiding the design of safer nanomaterials. Here we used a set of 17 stable suspensions of monodisperse amorphous silica nanoparticles (SNPs) with selected variations in size (diameter, 2-335 nm), surface area (BET, 16-422 m(2)/g) and microporosity (micropore volume, 0-71 microl/g) to assess with multiple regression analysis the physico-chemical determinants of the cytotoxic activity in four different cell types (J774 macrophages, EAHY926 endothelial cells, 3T3 fibroblasts and human erythrocytes). We found that the response to these SNPs is governed by different physico-chemical parameters which vary with cell type: In J774 macrophages, the cytotoxic activity (WST1 assay) increased with external surface area (alphas method) and decreased with micropore volume (r(2) of the model, 0.797); in EAHY926 and 3T3 cells, the cytotoxic activity of the SNPs (MTT and WST1 assay, respectively) increased with surface roughness and small diameter (r(2), 0.740 and 0.872, respectively); in erythrocytes, the hemolytic activity increased with the diameter of the SNP (r(2), 0.860). We conclude that it is possible to predict with good accuracy the in vitro cytotoxic potential of SNPs on the basis of their physico-chemical characteristics. These determinants are, however, complex and vary with cell type, reflecting the pleiotropic interactions of nanoparticles with biological systems.

Carbon black and titanium dioxide nanoparticles elicit distinct apoptotic pathways in bronchial epithelial cells.

BACKGROUND: Increasing environmental and occupational exposures to nanoparticles (NPs) warrant deeper insight into the toxicological mechanisms induced by these materials. The present study was designed to characterize the cell death induced by carbon black (CB) and titanium dioxide (TiO2) NPs in bronchial epithelial cells (16HBE14o- cell line and primary cells) and to investigate the implicated molecular pathways. RESULTS: Detailed time course studies revealed that both CB (13 nm) and TiO2(15 nm) NP exposed cells exhibit typical morphological (decreased cell size, membrane blebbing, peripheral chromatin condensation, apoptotic body formation) and biochemical (caspase activation and DNA fragmentation) features of apoptotic cell death. A decrease in mitochondrial membrane potential, activation of Bax and release of cytochrome c from mitochondria were only observed in case of CB NPs whereas lipid peroxidation, lysosomal membrane destabilization and cathepsin B release were observed during the apoptotic process induced by TiO2 NPs. Furthermore, ROS production was observed after exposure to CB and TiO2 but hydrogen peroxide (H2O2) production was only involved in apoptosis induction by CB NPs. CONCLUSIONS: Both CB and TiO2 NPs induce apoptotic cell death in bronchial epithelial cells. CB NPs induce apoptosis by a ROS dependent mitochondrial pathway whereas TiO2 NPs induce cell death through lysosomal membrane destabilization and lipid peroxidation. Although the final outcome is similar (apoptosis), the molecular pathways activated by NPs differ depending upon the chemical nature of the NPs.

The prevalence of hepatitis C virus (HCV) infection in HIV-positive individuals in the UK - trends in HCV testing and the impact of HCV on HIV treatment outcomes.

We examined the prevalence of hepatitis C virus (HCV) infection among HIV-positive individuals in the UK, trends in HCV testing and the impact of HCV on HIV treatment outcomes. Trends over time in HCV prevalence were calculated using each patient's most recent HCV status at the end of each calendar year. Logistic regression was used to identify factors associated with having a HCV antibody test, and Cox regression was used to determine whether HCV status was associated with the time to experiencing an immunological response to highly active antiretroviral treatment (HAART), time to virological response and viral rebound. Of the 31,765 HIV-positive individuals seen for care between January 1996 and September 2007, 20,365 (64.1%) individuals were tested for HCV, and 1807 (8.9%) had detectable HCV antibody. The proportion of patients in follow-up ever tested for HCV increased over time, from 782/8505 (9.2%) in 1996 to 14,280/17,872 (79.9%) in 2007. Nine thousand six hundred and sixty-nine individuals started HAART for the first time in or after January 2000, of whom, 396 (4.1%) were HCV positive. Presence of HCV infection did not affect initial virological response, virological rebound or immunological response. The cumulative prevalence of HCV in the UK CHIC Study is 8.9%. Despite UK guidelines, over 20% of HIV-positive individuals have not had their HCV status determined by 2007. HCV infection had no impact on HIV virological outcomes or immunological response to HIV treatment. The long-term impact on morbidity and mortality remain to be determined.

Oxidative stress and proinflammatory effects of carbon black and titanium dioxide nanoparticles: role of particle surface area and internalized amount.

The ubiquitous presence of nanoparticles (NPs) together with increasing evidence linking them to negative health effects points towards the need to develop the understanding of mechanisms by which they exert toxic effects. This study was designed to investigate the role of surface area and oxidative stress in the cellular effects of two chemically distinct NPs, carbon black (CB) and titanium dioxide (TiO(2)), on the bronchial epithelial cell line (16HBE14o-). CB and TiO(2) NPs were taken up by 16HBE cells in a dose-dependent manner and were localized within the endosomes or free in the cytoplasm. Oxidative stress produced inside the cell by NPs was well correlated to the BET surface area and endocytosis of NPs. Contrary to intracellular conditions only CB NPs produced reactive oxygen species (ROS) under abiotic conditions. Exposure of cells to NPs resulted in an increased granulocyte macrophage colony stimulating factor (GM-CSF) mRNA expression and secretion. Inflammatory effects of NPs were dependent on the surface area and were mediated through oxidative stress as they were inhibited by catalase. It can be concluded that NP induced oxidative stress and pro-inflammatory responses are well correlated not only with the BET (Brunauer, Emmett and Teller) surface of the individual NPs but also with the internalized amount of NPs. Differences of even few nanometers in primary particle size lead to significant changes in inflammatory and oxidative stress responses.

Intravitreal sustained-release ganciclovir implantation to control cytomegalovirus retinitis in AIDS.

A novel form of therapy, the intravitreal ganciclovir implant (Vitrasert), was used to treat cytomegalovirus (CMV) retinitis in AIDS, when it was not possible to continue systemic therapy or disease progression was evident despite conventional intravenous treatment. A review of the ophthalmic and general health data, operative records and fundus photographs of 9 individuals who had Vitrasert implantation surgery in Edinburgh. Two patients received bilateral replacement implant procedures. Visual acuity, concomitant anti-CMV therapy, complications of implantation and control of retinitis were documented until final follow-up. Control of CMV retinitis was achieved in all individuals at one month postoperatively and remained controlled for a mean period of 5.8 months. Postoperative complications were mild and transient in all but 2 eyes of 2 patients who developed non-progressive macula oedema. Blinding complications as a result of surgery were not encountered and retinal detachment was not observed in this series. Vitrasert implantation is an acceptable and well tolerated treatment for CMV retinitis when systemic therapy fails to control disease progression.

The molecular epidemiology of human immunodeficiency virus type 1 in six cities in Britain and Ireland.

We have sequenced the p17 coding regions of the gag gene from 211 patients infected either through injecting drug use (IDU) or by sexual intercourse between men from six cities in Scotland, N. England, N. Ireland, and the Republic of Ireland. All sequences were of subtype B. Phylogenetic analysis revealed substantial heterogeneity in the sequences from homosexual men. In contrast, sequence from over 80% of IDUs formed a relatively tight cluster, distinct both from those of published isolates and of the gay men. There was no large-scale clustering of sequences by city in either risk group, although a number of close associations between pairs of individuals were observed. From the known date of the HIV-1 epidemic among IDUs in Edinburgh, the rate of sequence divergence at synonymous sites is estimated to be about 0.8%. On this basis we estimate the date of divergence of the sequences among homosexual men to be about 1975, which may correspond to the origin of the B subtype epidemic.

Antimicrobial treatment of fish tank granuloma.

Three patients with fish tank granuloma of the hand and forearm are reported. Each patient was treated with antimicrobial regimes which have rarely or never been previously used in this condition. Two patients responded well to treatment, one who received ciprofloxacin plus clarithromycin and another who was given clarithromycin plus ethambutol. The third patient received six different antimicrobial regimes before responding to a combination of rifabutin and ciprofloxacin. Our experience suggests that there now exist a number of effective alternatives to antimicrobials which have been traditionally used in the treatment of cutaneous Mycobacterium marinum infection.

Clinical features, outcome and survival from cerebral toxoplasmosis in Edinburgh AIDS patients.

Nineteen cases of cerebral toxoplasmosis (CTOX) are reported from a group of Edinburgh AIDS patients. All patients were severely immunodeficient at the time of presentation with CD4 count < 50 cells/mm3. Thirteen patients had suffered a previous AIDS-defining illness. In Edinburgh, CTOX has developed in 48% of patients who are seropositive for toxoplasma and have a CD4 count < 50 cells/mm3. It is estimated that at least half of the toxoplasma seropositive patients will develop CTOX if they survive for 21 months after reaching a time in their illness when the CD4 count = 50 cells/mm3. The incidence of CTOX in toxoplasma-seronegative patients with a CD4 count < 50 cells/mm3 is 1.3%. All patients showed improvement on treatment and there was no correlation between clinical or radiological features and patient survival. Those patients unable to tolerate first choice anti-toxoplasma therapy had a significantly shorter survival than the remainder but there was no single therapeutic regimen which conferred a survival advantage. Eighteen patients had died at the time of study and the median survival following diagnosis of cerebral toxoplasmosis was 10 months (range 3-38 months). Postmortem examination of the brain was available in 8, 4 of whom had concomitant cerebral lymphoma. The survival from AIDS or CD4 count = 50 cells/mm3 did not differ significantly between those with treated CTOX and a control group who had no toxoplasma infection, suggesting that treatment is reasonably effective. CTOX is a disease associated with severe HIV-related immunodeficiency and, in those with a CD4 count < 50 cells/mm3, occurs more than 35 times as frequently in toxoplasma-seropositive than toxoplasma-seronegative patients. Treatment is effective but the outcome of treated disease cannot be predicted from presenting clinical or radiological features. Concomitant space-occupying cerebral pathology is evident in 50% of post-mortem examinations.