Development of a non-target strategy for evaluation of potential biological effects of inhalable aerosols generated during purposeful room conditioning using an in vitro inhalation model.

OBJECTIVES: An integrated in vitro inhalation approach was outlined to estimate potential adverse acute inhalation effects of aerosols from commercial nebulizer applications used for purposeful room conditioning such as disinfection, scenting or others. Aerosol characterization, exposure estimation and evaluation of acute biological effects by in vitro inhalation were included to generate dose-response data, allowing for determination of in vitro lowest observable adverse effect levels (LOAELs). Correlation of these to estimates of human lung deposition was included for quantitative in vitro to in vivo extrapolation approach (QIVIVE) for acute effects during human exposure. METHODS: To test the proposed approach, a case study was undertaken using two realistic test materials. An acute in vitro inhalation setup with air-liquid interface A549-cells in an optimized exposure situation (P.R.I.T.® ExpoCube®) was used to expose cells and analysis of relevant biological effects (viability, mitochondrial membrane potential, stress, IL-8 release) was carried out. RESULTS: The observed dose-responsive effects in a sub-toxic dose-range could be attributed to the main component of one test material and its presence in the aerosol phase of the nebulized material. QIVIVE resulted in a factor of at least 256 between the in vitro LOAEL and the estimated acute human lung exposure for this test material. CONCLUSIONS: The case-study shows the value of the non-target in vitro inhalation testing approach especially in case of a lack of knowledge on complex product composition. It is expected that approaches like this will be of high value for product safety and environmental health in the future.

Design of a routine in vitro inhalation approach to estimate biological effects of nebulized products.Application in a case study on a potential real product for purposeful room conditioning by use of a commercial nebulizer.Combining results from aerosol characterization and in vitro inhalation experiments allowed for comprehensive correlation of product composition, aerosol properties and biological effects.Assignment of sub-toxic biological effects to a specific product component enabled identification of a product composition with potentially even less biological effect.Combined in vivo exposure estimation and in vitro LOAEL determination enabled a QIVIVE approach.

Comparing in vitro human liver models to in vivo human liver using RNA-Seq.

The liver plays an important role in xenobiotic metabolism and represents a primary target for toxic substances. Many different in vitro cell models have been developed in the past decades. In this study, we used RNA-sequencing (RNA-Seq) to analyze the following human in vitro liver cell models in comparison to human liver tissue: cancer-derived cell lines (HepG2, HepaRG 3D), induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs), cancerous human liver-derived assays (hPCLiS, human precision cut liver slices), non-cancerous human liver-derived assays (PHH, primary human hepatocytes) and 3D liver microtissues. First, using CellNet, we analyzed whether these liver in vitro cell models were indeed classified as liver, based on their baseline expression profile and gene regulatory networks (GRN). More comprehensive analyses using non-differentially expressed genes (non-DEGs) and differential transcript usage (DTU) were applied to assess the coverage for important liver pathways. Through different analyses, we noticed that 3D liver microtissues exhibited a high similarity with in vivo liver, in terms of CellNet (C/T score: 0.98), non-DEGs (10,363) and pathway coverage (highest for 19 out of 20 liver specific pathways shown) at the beginning of the incubation period (0 h) followed by a decrease during long-term incubation for 168 and 336 h. PHH also showed a high degree of similarity with human liver tissue and allowed stable conditions for a short-term cultivation period of 24 h. Using the same metrics, HepG2 cells illustrated the lowest similarity (C/T: 0.51, non-DEGs: 5623, and pathways coverage: least for 7 out of 20) with human liver tissue. The HepG2 are widely used in hepatotoxicity studies, however, due to their lower similarity, they should be used with caution. HepaRG models, iPSC-HLCs, and hPCLiS ranged clearly behind microtissues and PHH but showed higher similarity to human liver tissue than HepG2 cells. In conclusion, this study offers a resource of RNA-Seq data of several biological replicates of human liver cell models in vitro compared to human liver tissue.

Cerium oxide and barium sulfate nanoparticle inhalation affects gene expression in alveolar epithelial cells type II.

BACKGROUND: Understanding the molecular mechanisms of nanomaterial interacting with cellular systems is important for appropriate risk assessment. The identification of early biomarkers for potential (sub-)chronic effects of nanoparticles provides a promising approach towards cost-intensive and animal consuming long-term studies. As part of a 90-day inhalation toxicity study with CeO2 NM-212 and BaSO4 NM-220 the present investigations on gene expression and immunohistochemistry should reveal details on underlying mechanisms of pulmonary effects. The role of alveolar epithelial cells type II (AEII cells) is focused since its contribution to defense against inhaled particles and potentially resulting adverse effects is assumed. Low dose levels should help to specify particle-related events, including inflammation and oxidative stress. RESULTS: Rats were exposed to clean air, 0.1, 0.3, 1.0, and 3.0 mg/m3 CeO2 NM-212 or 50.0 mg/m3 BaSO4 NM-220 and the expression of 391 genes was analyzed in AEII cells after one, 28 and 90 days exposure. A total number of 34 genes was regulated, most of them related to inflammatory mediators. Marked changes in gene expression were measured for Ccl2, Ccl7, Ccl17, Ccl22, Ccl3, Ccl4, Il-1α, Il-1ß, and Il-1rn (inflammation), Lpo and Noxo1 (oxidative stress), and Mmp12 (inflammation/lung cancer). Genes related to genotoxicity and apoptosis did not display marked regulation. Although gene expression was less affected by BaSO4 compared to CeO2 the gene pattern showed great overlap. Gene expression was further analyzed in liver and kidney tissue showing inflammatory responses in both organs and marked downregulation of oxidative stress related genes in the kidney. Increases in the amount of Ce were measured in liver but not in kidney tissue. Investigation of selected genes on protein level revealed increased Ccl2 in bronchoalveolar lavage of exposed animals and increased Lpo and Mmp12 in the alveolar epithelia. CONCLUSION: AEII cells contribute to CeO2 nanoparticle caused inflammatory and oxidative stress reactions in the respiratory tract by the release of related mediators. Effects of BaSO4 exposure are low. However, overlap between both substances were detected and support identification of potential early biomarkers for nanoparticle effects on the respiratory system. Signs for long-term effects need to be further evaluated by comparison to a respective exposure setting.

Biological effects of carbon black nanoparticles are changed by surface coating with polycyclic aromatic hydrocarbons.

BACKGROUND: Carbon black nanoparticles (CBNP) are mainly composed of carbon, with a small amount of other elements (including hydrogen and oxygen). The toxicity of CBNP has been attributed to their large surface area, and through adsorbing intrinsically toxic substances, such as polycyclic aromatic hydrocarbons (PAH). It is not clear whether a PAH surface coating changes the toxicological properties of CBNP by influencing their physicochemical properties, through the specific toxicity of the surface-bound PAH, or by a combination of both. METHODS: Printex®90 (P90) was used as CBNP; the comparators were P90 coated with either benzo[a]pyrene (BaP) or 9-nitroanthracene (9NA), and soot from acetylene combustion that bears various PAHs on the surface (AS-PAH). Oxidative stress and IL-8/KC mRNA expression were determined in A549 and bronchial epithelial cells (16HBE14o-, Calu-3), mouse intrapulmonary airways and tracheal epithelial cells. Overall toxicity was tested in a rat inhalation study according to Organization for Economic Co-operation and Development (OECD) criteria. Effects on cytochrome monooxygenase (Cyp) mRNA expression, cell viability and mucociliary clearance were determined in acute exposure models using explanted murine trachea. RESULTS: All particles had similar primary particle size, shape, hydrodynamic diameter and ζ-potential. All PAH-containing particles had a comparable specific surface area that was approximately one third that of P90. AS-PAH contained a mixture of PAH with expected higher toxicity than BaP or 9NA. PAH-coating reduced some effects of P90 such as IL-8 mRNA expression and oxidative stress in A549 cells, granulocyte influx in the in vivo OECD experiment, and agglomeration of P90 and mucus release in the murine trachea ex vivo. Furthermore, P90-BaP decreased particle transport speed compared to P90 at 10 µg/ml. In contrast, PAH-coating induced IL-8 mRNA expression in bronchial epithelial cell lines, and Cyp mRNA expression and apoptosis in tracheal epithelial cells. In line with the higher toxicity compared to P90-BaP and P90-9NA, AS-PAH had the strongest biological effects both ex vivo and in vivo. CONCLUSIONS: Our results demonstrate that the biological effect of CBNP is determined by a combination of specific surface area and surface-bound PAH, and varies in different target cells.

Toxicity profile of the GATA-3-specific DNAzyme hgd40 after inhalation exposure.

DNAzymes are single-stranded catalytic DNA molecules that bind and cleave specific sequences in a target mRNA molecule. Their potential as novel therapeutic agents has been demonstrated in a variety of disease models. However, no studies have yet addressed their toxicology and safety pharmacology profiles in detail. Here we describe a detailed toxicological analysis of inhaled hgd40, a GATA-3-specific DNAzyme designed for the treatment of allergic bronchial asthma. Subacute toxicity, immunotoxicity, and respiratory, cardiovascular, and CNS safety pharmacology were analyzed in rodents and non-rodents, and genotoxicity was assessed in human peripheral blood. Overall, hgd40 was very well tolerated when delivered by aerosol inhalation or slow intravenous infusion. Only marginal reversible histopathological changes were observed in the lungs of rats receiving the highest dose of inhaled hgd40. The changes consisted of slight mononuclear cell infiltration and alveolar histiocytosis, and moderate hyperplasia of bronchus-associated lymphoid tissue. No local or systemic adverse effects were observed in dogs. No compound-related respiratory, cardiovascular, or CNS adverse events were observed. The only relevant immunological findings were very slight dose-dependent changes in interleukin-10 and interferon-γ levels in bronchoalveolar lavage fluid. Taken together, these results support direct delivery of a DNAzyme via inhalation for the treatment of respiratory disease.

Relative oral bioavailability of glycidol from glycidyl fatty acid esters in rats.

In order to quantify the relative bioavailability of glycidol from glycidyl fatty acid esters in vivo, glycidyl palmitoyl ester and glycidol were orally applied to rats in equimolar doses. The time courses of the amounts of glycidol binding to hemoglobin as well as the excretion of 2,3-dihydroxypropyl mercapturic acids were determined. The results indicate that glycidol is released from the glycidyl ester by hydrolysis and rapidly distributed in the organism. In relation to glycidol, there was only a small timely delay in the binding to hemoglobin for the glycidol moiety released from the ester which may be certainly attributed to enzymatic hydrolysis. In both cases, however, an analogous plateau was observed representing similar amounts of hemoglobin binding. With regard to the urinary excretion of mercapturic acids, also similar amounts of dihydroxypropyl mercapturic acids could be detected. In an ADME test using a virtual double tag (³H, ¹4C) of glycidyl palmitoyl ester, a diverging isotope distribution was detected. The kinetics of the ¹4C-activity reflected the kinetics of free glycidol released after hydrolysis of the palmitoyl ester. In view of this experimental data obtained in rats, it is at present justified for the purpose of risk assessment to assume complete hydrolysis of the glycidyl ester in the gastrointestinal tract. Therefore, assessment of human exposure to glycidyl fatty acid ester should be regarded as an exposure to the same molar quantity of glycidol.

Lung alterations following single or multiple low-dose carbon black nanoparticle aspirations in mice.

Carbon black nanoparticle (CBNP) applications in high doses have been shown to be harmful to the lung. It is postulated that even small, environmentally relevant concentrations induce changes on lung homeostasis. The present study determined the impact of low-dose single and multiple CBNP (Printex 90) applications on mouse alveolar cell metabolism, especially inflammatory and oxidative stress parameters. Nanoparticles were administered to mice by a single or 8 oropharyngeal aspirations at wk 1, 2, 3, 5, 7, 9, 11, and 12 using 7 µg Printex 90, 7 µg DQ12 quartz (positive control), with water vehicle and saline as negative controls. After 2 d or 3 mo lung function was analyzed. Further lung histology, bronchoalveolar lavage fluid (BALF) parameters, and mRNA expression of cytokines and antioxidants enzymes in type II pneumocytes were measured on d 3 or after 3 mo. Single low-dose Printex 90 application induced no marked alterations in lung functions or BALF phospholipid levels but significant decrease in superoxide dismutase 2 and numerically elevated glutathione peroxidase 3 mRNA expression levels in type II pneumocytes. Multiple CBNP applications produced reduced lung function, collagen accumulation, elevated phospholipid levels in BALF, and a massive infiltration of macrophages. Type II pneumocyte mRNA expression of antioxidative enzymes remained unchanged throughout the subchronic experiment, but showed a significant decrease in interleukin (IL)-6Rα mRNA expression. This study demonstrates that an environmentally relevant CBNP concentration induced an acute inflammatory response, an effect that is exacerbated throughout the subchronic duration.

Pulmonary toxicity of printer toner following inhalation and intratracheal instillation.

The pulmonary effects of a finished toner were evaluated in intratracheal instillation and inhalation studies, using toners with external additives (titanium dioxide nanoparticles and amorphous silica nanoparticles). Rats received an intratracheal dose of 1 mg or 2 mg of toner and were sacrificed at 3 days, 1 week, 1 month, 3 months and 6 months. The toner induced pulmonary inflammation, as evidenced by a transient neutrophil response in the low-dose groups and persistent neutrophil infiltration in the high-dose groups. There were increased concentrations of heme oxygenase-1 (HO-1) as a marker of oxidative stress in the bronchoalveolar lavage fluid (BALF) and the lung. In a 90-day inhalation study, rats were exposed to well-dispersed toner (mean of MMAD: 3.76 µm). The three mass concentrations of toner were 1, 4 and 16 mg/m(3) for 13 weeks, and the rats were sacrificed at 6 days and 91 days after the end of the exposure period. The low and medium concentrations did not induce neutrophil infiltration in the lung of statistical significance, but the high concentration did, and, in addition, upon histopathological examination not only showed findings of inflammation but also of fibrosis in the lung. Taken together, the results of our studies suggest that toners with external additives lead to pulmonary inflammation and fibrosis at lung burdens suggest beyond the overload. The changes observed in the pulmonary responses in this inhalation study indicate that the high concentration (16 mg/m(3)) is an LOAEL and that the medium concentration (4 mg/m(3)) is an NOAEL.

Photosensitivity and filter efficacy in albinism✰.

PURPOSE: To describe the prevalence and severity of photosensitivity in patients with albinism, and to compare with ocular features and how this correlated with use and choice of optical filters. METHODS: Cross-sectional study on 81 participants with ocular or oculocutaneous albinism. An ophthalmic evaluation including visual acuity, contrast sensitivity and evaluation of iris translucency and fundus hypopigmentation was performed. Participants were offered optical rehabilitation with testing of a wide panel of filters. The associations between ocular characteristics, subjective photosensitivity complaints, and filter choice were evaluated. RESULTS: Photosensitivity was rated as "some" to "worst imaginable" in 77.8% of participants. Severity of photosensitivity correlated significantly with fundus hypopigmentation (p = 0.04) but not with iris translucency (p = 0.14) and it was worse in those with poor visual acuity but there was no association between photosensitivity and contrast vision. Seventy-four new pairs of spectacles were prescribed in the study. All outdoor spectacles contained a filter, whereas 26.5% of new indoor spectacles did not. Relatively neutral filter colors (gray, brown or a combination of gray and brown with other colors) and low transmission were preferred. DISCUSSION: Photosensitivity is common in albinism, but research targeting treatment is limited. Color and neutral filters with a low light transmission were preferred, with participants having a large number of spectacles, presumably to meet their needs in different situations.

An improved guideline adherence and PPI efficacy has been accompanied by a decrease in diagnostic delay, and strictures before diagnosis of eosinophilic esophagitis in the North Denmark Region - a retrospective registry study of the DanEoE cohorts.

BACKGROUND: In the North Denmark Region an increased awareness of eosinophilic esophagitis (EoE) was observed after 2011 where a regional biopsy guideline was implemented. This resulted in an increased awareness of EoE and a 50-fold increase in the incidence of EoE patients between 2007-2017. AIMS: The aims of this study were to examine the progress in diagnostic delay, complications, PPI treatment, and follow up since 2017 in Danish patients with eosinophilic esophagitis. MATERIALS AND METHODS: This was a retrospective registry- and population-based cohort study (DanEoE2 cohort) including 346 adult patients with esophageal eosinophilia diagnosed between 2018-2021 in the North Denmark Region. The DanEoE2 cohort included all possible EoE patients by using the Danish Patho-histology registry based on the SNOMED-system. The data was analyzed and compared to the DanEoE cohort (2007-2017). RESULTS: The diagnostic delay of EoE patients diagnosed between 2018-2021 in the North Denmark Region had decreased with a median of 1.5 years (5.5 (2.0;12) years versus 4.0 (1.0;12) years, p=0.03). Strictures before diagnosis had decreased 8.4 % (11.6% versus 3.2%, p=0.003). The number of patients started on high-dose PPI increased (56% versus 88%, p<0.001). An intensified awareness regarding national guidelines and follow-up was observed as an increase in the number of histological follow up (67% versus 74%, p=0.05). CONCLUSIONS: Comparisons of the DanEoE cohorts showed a decrease in diagnostic delay, a decrease in stricture formation before diagnosis, and an improved guideline adherence after 2017. Future studies are needed to assess if symptomatic or histological remission on PPI treatment is more capable of predicting a patient's risk of developing complications.

Occupational exposure to veterinary antibiotics: Pharmacokinetics of enrofloxacin in humans after dermal, inhalation and oral uptake - A clinical study.

Occupational exposure to veterinary antibiotics in hen houses at poultry feeding farms was demonstrated by biomonitoring campaigns in the past. The objective of this study was to investigate pharmacokinetics of three uptake routes: dermal, oral and inhaled. In an open-label cross-over study, six healthy volunteers were exposed to single occupational relevant doses of enrofloxacin. Plasma and urine samples were analysed for enrofloxacin and ciprofloxacin. Physiologically based pharmacokinetic (PBPK) modelling based on bioanalysis data showed underestimation for the elimination rate in comparison to experimental data pointing towards a lack of sufficient ADME information and limitations of available physico-chemical properties of the parent drug. The data obtained in this study indicate that oral uptake with its various sources, e.g. airborne enrofloxacin, direct hand-mouth contact, is the major source for occupational exposure to enrofloxacin in hen houses. Dermal exposure was considered negligible.

Substantiate a read-across hypothesis by using transcriptome data-A case study on volatile diketones.

This case study explores the applicability of transcriptome data to characterize a common mechanism of action within groups of short-chain aliphatic α-, ß-, and γ-diketones. Human reference in vivo data indicate that the α-diketone diacetyl induces bronchiolitis obliterans in workers involved in the preparation of microwave popcorn. The other three α-diketones induced inflammatory responses in preclinical in vivo animal studies, whereas beta and gamma diketones in addition caused neuronal effects. We investigated early transcriptional responses in primary human bronchiolar (PBEC) cell cultures after 24 h and 72 h of air-liquid exposure. Differentially expressed genes (DEGs) were assessed based on transcriptome data generated with the EUToxRisk gene panel of Temp-O-Seq®. For each individual substance, genes were identified displaying a consistent differential expression across dose and exposure duration. The log fold change values of the DEG profiles indicate that α- and ß-diketones are more active compared to γ-diketones. α-diketones in particular showed a highly concordant expression pattern, which may serve as a first indication of the shared mode of action. In order to gain a better mechanistic understanding, the resultant DEGs were submitted to a pathway analysis using ConsensusPathDB. The four α-diketones showed very similar results with regard to the number of activated and shared pathways. Overall, the number of signaling pathways decreased from α-to ß-to γ-diketones. Additionally, we reconstructed networks of genes that interact with one another and are associated with different adverse outcomes such as fibrosis, inflammation or apoptosis using the TRANSPATH-database. Transcription factor enrichment and upstream analyses with the geneXplain platform revealed highly interacting gene products (called master regulators, MRs) per case study compound. The mapping of the resultant MRs on the reconstructed networks, visualized similar gene regulation with regard to fibrosis, inflammation and apoptosis. This analysis showed that transcriptome data can strengthen the similarity assessment of compounds, which is of particular importance, e.g., in read-across approaches. It is one important step towards grouping of compounds based on biological profiles.

Impacts after inhalation of nano- and fine-sized titanium dioxide particles: morphological changes, translocation within the rat lung, and evaluation of particle deposition using the relative deposition index.

A 3-week inhalation study with nano- and fine-sized titanium dioxide (TiO(2)) with 3, 28, and 90 days recovery time was performed in female Wistar rats. Lung volume measurements, histology, electron microscopy, hematology, and bronchoalveolar lavage (BAL) fluid analyses were conducted and the relative deposition index (RDI) was calculated. Minimal inflammatory changes in the lungs, leucopenia, and a decrease in ß-glucuronidase were observed. Particles were mainly deposited in alveolar macrophages and, to a lesser extent, in type-I pneumocytes, and this was quantified using the RDI. Rarely, particle-laden cells were observed inside capillaries. Therefore, minimal translocation of particles into the bloodstream has to be considered. Significant changes, e.g. in elicited effects or translocation behavior, between nano- and fine-particle-treated groups were not observed.

Hepatocellular Carcinoma Is a Natural Target for Adeno-Associated Virus (AAV) 2 Vectors.

Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current-or develop novel-strategies for treating HCC.

A modified protocol for successful miRNA profiling in human precision-cut lung slices (PCLS).

OBJECTIVE: Human precision cut lung slices (PCLS) are widely used as an ex vivo model system for drug discovery and development of new therapies. PCLS reflect the functional heterogeneity of lung tissue and possess relevant lung cell types. We thus determined the use of PCLS in studying non-coding RNAs notably miRNAs, which are important gene regulatory molecules. Since miRNAs play key role as mediators of respiratory diseases, they can serve as valuable prognostic or diagnostic biomarkers, and in therapeutic interventions, of lung diseases. A technical limitation though is the vast amount of agarose in PCLS which impedes (mi)RNA extraction by standard procedures. Here we modified our recently published protocol for RNA isolation from PCLS to enable miRNA readouts. RESULTS: The modified method relies on the separation of lysis and precipitation steps, and a clean-up procedure with specific magnetic beads. We obtained successfully quality miRNA amenable for downstream applications such as RTqPCR and whole transcriptome miRNA analysis. Comparison of miRNA profiles in PCLS with published data from human lung, identified all important miRNAs regulated in IPF, COPD, asthma or lung cancer. Therefore, this shows suitability of the method for analyzing miRNA targets and biomarkers in the valuable human PCLS model.

Genotoxicity testing of sulfur dioxide (SO2) in a mouse bone marrow micronucleus test complemented with hematological endpoints.

Sulfur dioxide (SO2) is a non-flammable, non-explosive, colorless gas. It is a ubiquitous environmental pollutant and an important chemical intermediate in several industrial processes. The toxicological properties of SO2, including its genotoxic potential, have been studied extensively. The majority of the available in vitro data indicate a lack of genotoxicity of SO2, while for sulfite salts some positive results have been reported. However, recent in vivo studies, using Kunming albino mice, have pointed to in vivo clastogenicity of SO2. To re-evaluate these positive findings, a bone-marrow micronucleus test according to OECD Guideline No. 474 was performed. NMRI mice (m/f) were exposed by inhalation via whole-body exposure to 0 (clean air), 2.7, 8, 27, or 80mg/m3 (0, 1, 3, 10, or 30ppm) SO2 for 4h/day on 7 consecutive days. Animals were sacrificed 24h after start of the last exposure, and blood samples (for complementing hematology) and bone marrow smears (for analysis of micronuclei) were prepared. Under the conditions used, exposure to SO2 caused no acute toxicity, mortality, or reduction in body weight. Compared with the clean-air controls, hematological parameters such as hematocrit, hemoglobin, erythrocyte/platelet/total leukocyte counts, differential white blood cell counts, and indicators of blood formation (reticulocyte counts, ratio of polychromatic to normochromatic erythrocytes in the bone marrow) remained unchanged by SO2 treatment. Unlike the previously reported studies on micronucleus formation, SO2 did not induce micronuclei in polychromatic erythrocytes of the bone marrow, whereas the positive control cyclophosphamide (60mg/kg body weight) was quite effective in this respect. Interestingly, SO2 treatment significantly enhanced malondialdehyde levels in erythrocyte lysates (TBARS method), indicating SO2-mediated oxidative stress, but also demonstrating systemic availability of the inhaled SO2. In conclusion, the present study could not reproduce the genotoxicity findings of the previously reported studies. SO2 is thus considered non-genotoxic in polychromatic erythrocytes in the bone marrow of NMRI mice under the conditions and in the concentrations used.

In vitro inhalation cytotoxicity testing of therapeutic nanosystems for pulmonary infection.

Due to the increasing need of new treatment options against bacterial lung infections, novel antimicrobial peptides (AMPs) are under development. Local bioavailability and less systemic exposure lead to the inhalation route of administration. Combining AMPs with nanocarriers (NCs) into nanosystems (NSs) might be a technique for improved results. An air-liquid interface (ALI) in vitro inhalation model was set up including a human alveolar lung cell line (A549) and an optimized exposure system (P.R.I.T.® ExpoCube®) to predict acute local lung toxicity. The approach including aerosol controls (cupper-II-sulfate and lactose) delivered lowest observable adverse effect levels (LOAELs). Different combinations of AMPs (AA139, M33) and NCs (polymeric nanoparticles (PNPs), micelles and liposomes) were tested under ALI and submerged in vitro conditions. Depending on the nature of AMP and NCs, packing of AMPs into NSs reduced the AMP-related toxicity. Large differences were found between the LOAELs determined by submerged or ALI testing with the ALI approach indicating higher sensitivity of the ALI model. Since aerosol droplet exposure is in vivo relevant, it is assumed that ALI based results represents the more significant source than submerged testing for in vivo prediction of local acute lung toxicity. In accordance with the current state-of-the-art view, this study shows that ALI in vitro inhalation models are promising tools to further develop in vitro methods in the field of inhalation toxicology.

Surface defects reduce Carbon Nanotube toxicity in vitro.

The cytotoxicity of two different types of Multi-walled Carbon Nanotubes (MWCNTs) in A549 lung epithelial cells and HepG2 hepatocytes was investigated. One MWCNT still contained iron that was used as a catalyst during production, while the other one had all iron removed in a post-production heat treatment resulting in significantly fewer surface defects. The WST-8 assay was applied to test cell viability. To check the integrity of the cell membrane, we performed the lactate dehydrogenases assay (LDH) and measured the cellular production of reactive oxygen species (ROS). Finally, to examine cell proliferation, we conducted a cell cycle analysis. The results showed a dose- and time-dependent decrease in cell viability for both MWCNTs in both cell types. Moreover, a dose- and time-dependent increase in LDH leakage was detected, thereby indicating a decreased membrane integrity. The production of ROS was significantly increased in the case of the heat-treated MWCNTs. The heat-treated MWCNTs showed significantly stronger adverse effects when compared to the non-treated MWCNTs. Additionally, the heat-treated MWCNTs induced a dose-dependent cell cycle arrest in A549 cells. Both MWCNTs induced a significant cytotoxicity, whereby the heat treatment, leading to a decrease in surface defects, further increased the indicated adverse effects.

Infliximab for inflammatory bowel disease in Denmark 1999-2005: clinical outcome and follow-up evaluation of malignancy and mortality.

BACKGROUND & AIMS: Data on safety and long-term follow-up evaluation of population-based cohorts of inflammatory bowel disease (IBD) patients treated with infliximab are sparse. The aim of this article is to describe the use of infliximab in a national Danish population-based IBD cohort during 1999-2005. METHODS: Medical records of all infliximab-treated IBD patients were scrutinized to abstract information on patient demographics, treatment efficacy, and adverse events. RESULTS: A total of 651 patients (619 with Crohn's disease, 15 with ulcerative colitis, and 17 with colonic IBD type unclassified) received infliximab during 1999-2005. A total of 3351 infusions were administered, with a median of 3 infusions per patient. A positive clinical response was observed in 82.7% (95% confidence interval, 79.9-85.5) of patients. Infusion reactions were observed after 146 of 3351 infusions (4.4%). Significantly fewer infusion reactions were seen in patients also receiving azathioprine or methotrexate (63 of 2079; 3.0%), compared with patients not receiving azathioprine or methotrexate (83 of 1272; 6.5%) (P < .0001). Severe adverse events were observed after 112 of 3351 infusions (3.3%) in a total of 95 patients (14.6%). Four patients developed cancer versus 5.9 expected (standardized incidence ratio, 0.7; 95 confidence interval, 0.2-1.7) and 13 patients died versus 6.9 expected (standardized mortality ratio, 1.9; 95% confidence interval, 1.0-3.2). Two deaths caused by infections were possibly related to infliximab. CONCLUSIONS: Infliximab seemed effective in IBD and generally was well tolerated. However, rare but severe adverse events occurred, and patients receiving infliximab therefore should be selected carefully and monitored closely. No lymphomas and no increased risk of cancer were observed.