Malignant peritoneal mesotheliomas of rats induced by multiwalled carbon nanotubes and amosite asbestos: transcriptome and epigenetic profiles.

BACKGROUND: Malignant mesothelioma is an aggressive cancer that often originates in the pleural and peritoneal mesothelium. Exposure to asbestos is a frequent cause. However, studies in rodents have shown that certain multiwalled carbon nanotubes (MWCNTs) can also induce malignant mesothelioma. The exact mechanisms are still unclear. To gain further insights into molecular pathways leading to carcinogenesis, we analyzed tumors in Wistar rats induced by intraperitoneal application of MWCNTs and amosite asbestos. Using transcriptomic and epigenetic approaches, we compared the tumors by inducer (MWCNTs or amosite asbestos) or by tumor type (sarcomatoid, epithelioid, or biphasic). RESULTS: Genome-wide transcriptome datasets, whether grouped by inducer or tumor type, showed a high number of significant differentially expressed genes (DEGs) relative to control peritoneal tissues. Bioinformatic evaluations using Ingenuity Pathway Analysis (IPA) revealed that while the transcriptome datasets shared commonalities, they also showed differences in DEGs, regulated canonical pathways, and affected molecular functions. In all datasets, among highly- scoring predicted canonical pathways were Phagosome Formation, IL8 Signaling, Integrin Signaling, RAC Signaling, and TREM1 Signaling. Top-scoring activated molecular functions included cell movement, invasion of cells, migration of cells, cell transformation, and metastasis. Notably, we found many genes associated with malignant mesothelioma in humans, which showed similar expression changes in the rat tumor transcriptome datasets. Furthermore, RT-qPCR revealed downregulation of Hrasls, Nr4a1, Fgfr4, and Ret or upregulation of Rnd3 and Gadd45b in all or most of the 36 tumors analyzed. Bisulfite sequencing of Hrasls, Nr4a1, Fgfr4, and Ret revealed heterogeneity in DNA methylation of promoter regions. However, higher methylation percentages were observed in some tumors compared to control tissues. Lastly, global 5mC DNA, m6A RNA and 5mC RNA methylation levels were also higher in tumors than in control tissues. CONCLUSIONS: Our findings may help better understand how exposure to MWCNTs can lead to carcinogenesis. This information is valuable for risk assessment and in the development of safe-by-design strategies.

Immunohistochemical Characterization of Proliferative Lesions in the Thymus of Aging CD-1 Mice From Two Studies on the RITA Database, With Special Reference to the Perivascular Space.

Thymic lymphoid hyperplasia is a common age-related finding, which occurs particularly in female CD-1 mice. The main differential diagnoses are malignant lymphoma and thymoma. A systematic investigation of control groups from two carcinogenicity studies was performed including measurements of thymic size, and the immunohistochemistry (IHC) markers pan-Cytokeratin (pan-CK) for thymic epithelial cells; CD3 and CD45R/B220 for T and B lymphocytes, respectively; CD31 for endothelial cells; and F4/80 for macrophages. Thymoma can be differentiated by increased numbers of proliferating epithelial cells demonstrated by pan-CK IHC staining. Differentiation between lymphoid hyperplasia and lymphoma is more challenging as a mixture of B and T lymphocytes can be present in both findings. The present investigation showed that the thymic perivascular space is the compartment where the increased numbers of lymphocytes in hyperplasia are localized and not the medulla, as previously thought. The lymphoepithelial compartment is atrophic to the same extent in thymi diagnosed with age-related involution or lymphoid hyperplasia. Both diagnoses are thus related to variations in lymphoid cellularity of the nonepithelial perivascular space, which is continuous with the perithymic tissue. Likewise, lymphomas have a predilection to colonize the perivascular space and to spare the lymphoepithelial compartment.

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Nonproliferative and Proliferative Lesions of the Minipig.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions) Project (www.toxpath.org/inhand.asp) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in most tissues and organs from the minipig used in nonclinical safety studies. Some of the lesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions as well as lesions induced by exposure to test materials. Relevant infectious and parasitic lesions are included as well. A widely accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common language among regulatory and scientific research organizations in different countries and increase and enrich international exchanges of information among toxicologists and pathologists.

BAL Cell Gene Expression Is Indicative of Outcome and Airway Basal Cell Involvement in Idiopathic Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a fatal disease with a variable and unpredictable course. OBJECTIVES: To determine whether BAL cell gene expression is predictive of survival in IPF. METHODS: This retrospective study analyzed the BAL transcriptome of three independent IPF cohorts: Freiburg (Germany), Siena (Italy), and Leuven (Belgium) including 212 patients. BAL cells from 20 healthy volunteers, 26 patients with sarcoidosis stage III and IV, and 29 patients with chronic obstructive pulmonary disease were used as control subjects. Survival analysis was performed by Cox models and component-wise boosting. Presence of airway basal cells was tested by immunohistochemistry and flow cytometry. MEASUREMENTS AND MAIN RESULTS: A total of 1,582 genes were predictive of mortality in the IPF derivation cohort in univariate analyses adjusted for age and sex at false discovery rate less than 0.05. A nine-gene signature, derived from the discovery cohort (Freiburg), performed well in both replication cohorts, Siena (P < 0.0032) and Leuven (P = 0.0033). nCounter expression analysis confirmed the array results (P < 0.0001). The genes associated with mortality in BAL cells were significantly enriched for genes expressed in airway basal cells. Further analyses by gene expression, flow cytometry, and immunohistochemistry showed an increase in airway basal cells in BAL and tissues of IPF compared with control subjects, but not in chronic obstructive pulmonary disease or sarcoidosis. CONCLUSIONS: Our results identify and validate a BAL signature that predicts mortality in IPF and improves the accuracy of outcome prediction based on clinical parameters. The BAL signature associated with mortality unmasks a potential role for airway basal cells in IPF.

Prenatal treatment with rosiglitazone attenuates vascular remodeling and pulmonary monocyte influx in experimental congenital diaphragmatic hernia.

INTRODUCTION: Extensive vascular remodeling causing pulmonary hypertension (PH) represents a major cause of mortality in patients with congenital diaphragmatic hernia (CDH). The chemokine monocyte chemoattractant protein-1 (MCP-1) is a biomarker for the severity of PH and its activation is accompanied by pulmonary influx of monocytes and extensive vascular remodeling. MCP-1 activation can be reversed by application of rosiglitazone (thiazolidinedione). We performed this study to evaluate the role of MCP-1 for the pathogenesis of PH in experimental CDH. We hypothesized that vascular remodeling and MCP-1 activation is accompanied by pulmonary influx of fetal monocytes and can be attenuated by prenatal treatment with rosiglitazone. METHODS: In a first set of experiments pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were harvested on D21 and divided into CDH and control. Quantitative real-time polymerase chain reaction, Western blot (WB), and immunohistochemistry (IHC) were used to evaluate MCP-1 expression, activation, and localization. Quantification and localization of pulmonary monocytes/macrophages were carried out by IHC. In a second set of experiments nitrofen-exposed dams were randomly assigned to prenatal treatment with rosiglitazone or placebo on D18+D19. Fetal lungs were harvested on D21, divided into control, CDH+rosiglitazone, and CDH+placebo and evaluated by WB as well as IHC. RESULTS: Increased thickness of pulmonary arteries of CDH fetuses was accompanied by increased systemic and perivascular MCP-1 protein expression and significantly higher amounts of pulmonary monocytes/macrophages compared to controls (p<0.01). These effects were reversed by prenatal treatment with rosiglitazone (p<0.01 vs. CDH+P; control). CONCLUSION: Prenatal treatment with rosiglitazone has the potential to attenuate activation of pulmonary MCP-1, pulmonary monocyte influx, and vascular remodeling in experimental CDH. These results provide a basis for future research on prenatal immunomodulation as a novel treatment strategy to decrease secondary effects of PH in CDH.

Human Teratoma-Derived Hematopoiesis Is a Highly Polyclonal Process Supported by Human Umbilical Vein Endothelial Cells.

Hematopoietic stem cells (HSCs) ensure a life-long regeneration of the blood system and are therefore an important source for transplantation and gene therapy. The teratoma environment supports the complex development of functional HSCs from human pluripotent stem cells, which is difficult to recapitulate in culture. This model mimics various aspects of early hematopoiesis, but is restricted by the low spontaneous hematopoiesis rate. In this study, a feasible protocol for robust hematopoiesis has been elaborated. We achieved a significant increase of the teratoma-derived hematopoietic population when teratomas were generated in the NSGS mouse, which provides human cytokines, together with co-injection of human umbilical vein endothelial cells. Since little is known about hematopoiesis in teratomas, we addressed localization and clonality of the hematopoietic lineage. Our results indicate that early human hematopoiesis is closely reflected in teratoma formation, and thus highlight the value of this model.

Isolated aggregates of lymphoid cells in the inner bronchial wall in asthma patients.

Here, we report findings in volunteers with bronchial asthma. Biopsies were obtained from the inner bronchial wall before and a short time again after segmental allergen provocation. In most of the baseline biopsies and in all evaluable biopsies after segmental allergen provocation, the follicular lymphoid tissue was detected by immunohistochemistry in the epithelium of these asthmatic patients. The basic occurrence of the tertiary lymphoid tissue in the bronchial mucosa of mild asthmatics was unexpected and may have consequences for the interpretation of pathophysiology, e.g., as a cause or consequence of bronchial asthma.

Nonproliferative and Proliferative Lesions of the Rat and Mouse Endocrine System.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Project (www.toxpath.org/inhand.asp) is a joint initiative among the Societies of Toxicological Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the endocrine organs (pituitary gland, pineal gland, thyroid gland, parathyroid glands, adrenal glands and pancreatic islets) of laboratory rats and mice, with color photomicrographs illustrating examples of the lesions. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for endocrine lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Human Effector Memory T Helper Cells Engage with Mouse Macrophages and Cause Graft-versus-Host-Like Pathology in Skin of Humanized Mice Used in a Nonclinical Immunization Study.

Humanized mice engrafted with human hematopoietic stem cells and developing functional human T-cell adaptive responses are in critical demand to test human-specific therapeutics. We previously showed that humanized mice immunized with long-lived induced-dendritic cells loaded with the pp65 viral antigen (iDCpp65) exhibited a faster development and maturation of T cells. Herein, we evaluated these effects in a long-term (36 weeks) nonclinical model using two stem cell donors to assess efficacy and safety. Relative to baseline, iDCpp65 immunization boosted the output of effector memory CD4+ T cells in peripheral blood and lymph nodes. No weight loss, human malignancies, or systemic graft-versus-host (GVH) disease were observed. However, for one reconstitution cohort, some mice immunized with iDCpp65 showed GVH-like signs on the skin. Histopathology analyses of the inflamed skin revealed intrafollicular and perifollicular human CD4+ cells near F4/80+ mouse macrophages around hair follicles. In spleen, CD4+ cells formed large clusters surrounded by mouse macrophages. In plasma, high levels of human T helper 2-type inflammatory cytokines were detectable, which activated in vitro the STAT5 pathway of murine macrophages. Despite this inflammatory pattern, human CD8+ T cells from mice with GVH reacted against the pp65 antigen in vitro. These results uncover a dynamic cross-species interaction between human memory T cells and mouse macrophages in the skin and lymphatic tissues of humanized mice.

Massive Clonal Selection and Transiently Contributing Clones During Expansion of Mesenchymal Stem Cell Cultures Revealed by Lentiviral RGB-Barcode Technology.

UNLABELLED: Mesenchymal stem (or stromal) cells (MSCs) have been used in more than 400 clinical trials for the treatment of various diseases. The clinical benefit and reproducibility of results, however, remain extremely variable. During the in vitro expansion phase, which is necessary to achieve clinically relevant cell numbers, MSCs show signs of aging accompanied by different contributions of single clones to the mass culture. Here we used multicolor lentiviral barcode labeling to follow the clonal dynamics during in vitro MSC expansion from whole umbilical cord pieces (UCPs). The clonal composition was analyzed by a combination of flow cytometry, fluorescence microscopy, and deep sequencing. Starting with highly complex cell populations, we observed a massive reduction in diversity, transiently dominating populations, and a selection of single clones over time. Importantly, the first wave of clonal constriction already occurred in the early passages during MSC expansion. Consecutive MSC cultures from the same UCP implied the existence of more primitive, MSC culture-initiating cells. Our results show that microscopically homogenous MSC mass cultures consist of many subpopulations, which undergo clonal selection and have different capabilities. Among other factors, the clonal composition of the graft might have an impact on the functional properties of MSCs in experimental and clinical settings. SIGNIFICANCE: Mesenchymal stem cells (MSCs) can easily be obtained from various adult or embryonal tissues and are frequently used in clinical trials. For their clinical application, MSCs have to be expanded in vitro. This unavoidable step influences the features of MSCs, so that clinical benefit and experimental results are often highly variable. Despite a homogenous appearance under the microscope, MSC cultures undergo massive clonal selection over time. Multicolor fluorescence labeling and deep sequencing were used to demonstrate the dynamic clonal composition of MSC cultures, which might ultimately explain the variable clinical performance of the cells.

Recommendations from the INHAND Apoptosis/Necrosis Working Group.

Historically, there has been confusion relating to the diagnostic nomenclature for individual cell death. Toxicologic pathologists have generally used the terms "single cell necrosis" and "apoptosis" interchangeably. Increased research on the mechanisms of cell death in recent years has led to the understanding that apoptosis and necrosis involve different cellular pathways and that these differences can have important implications when considering overall mechanisms of toxicity, and, for these reasons, the separate terms of apoptosis and necrosis should be used whenever differentiation is possible. However, it is also recognized that differentiation of the precise pathway of cell death may not be important, necessary, or possible in routine toxicity studies and so a more general term to indicate cell death is warranted in these situations. Morphological distinction between these two forms of cell death can sometimes be straightforward but can also be challenging. This article provides a brief discussion of the cellular mechanisms and morphological features of apoptosis and necrosis as well as guidance on when the pathologist should use these terms. It provides recommended nomenclature along with diagnostic criteria (in hematoxylin and eosin [H&E]-stained sections) for the most common forms of cell death (apoptosis and necrosis). This document is intended to serve as current guidance for the nomenclature of cell death for the International Harmonization of Nomenclature and Diagnostic Criteria Organ Working Groups and the toxicologic pathology community at large. The specific recommendations are:Use necrosis and apoptosis as separate diagnostic terms.Use modifiers to denote the distribution of necrosis (e.g., necrosis, single cell; necrosis, focal; necrosis, diffuse; etc.).Use the combined term apoptosis/single cell necrosis whenThere is no requirement or need to split the processes, orWhen the nature of cell death cannot be determined with certainty, orWhen both processes are present together. The diagnosis should be based primarily on the morphological features in H&E-stained sections. When needed, additional, special techniques to identify and characterize apoptosis can also be used.

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) progress to date and future plans.

The INHAND Proposal (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) has been operational since 2005. A Global Editorial Steering Committee (GESC) manages the overall objectives of the project and the development of harmonized terminology for each organ system is the responsibility of the Organ Working Groups (OWG), drawing upon experts from North America, Europe and Japan.Great progress has been made with 9 systems published to date - Respiratory, Hepatobiliary, Urinary, Central/Peripheral Nervous Systems, Male Reproductive and Mammary, Zymbals, Clitoral and Preputial Glands in Toxicologic Pathology and the Integument and Soft Tissue and Female Reproductive System in the Journal of Toxicologic Pathology as supplements and on a web site - www.goreni.org. INHAND nomenclature guides offer diagnostic criteria and guidelines for recording lesions observed in rodent toxicity and carcinogenicity studies. The guides provide representative photo-micrographs of morphologic changes, information regarding pathogenesis, and key references. During 2012, INHAND GESC representatives attended meetings with representatives of the FDA Center for Drug Evaluation and Research (CDER), Clinical Data Interchange Standards Consortium (CDISC), and the National Cancer Institute (NCI) Enterprise Vocabulary Services (EVS) to begin incorporation of INHAND terminology as preferred terminology for SEND (Standard for Exchange of Nonclinical Data) submissions to the FDA. The interest in utilizing the INHAND nomenclature, based on input from industry and government toxicologists as well as information technology specialists, suggests that there will be wide acceptance of this nomenclature. The purpose of this publication is to provide an update on the progress of INHAND.

The carcinogenic effect of various multi-walled carbon nanotubes (MWCNTs) after intraperitoneal injection in rats.

BACKGROUND: Biological effects of tailor-made multi-walled carbon nanotubes (MWCNTs) without functionalization were investigated in vivo in a two-year carcinogenicity study. In the past, intraperitoneal carcinogenicity studies in rats using biopersistent granular dusts had always been negative, whereas a number of such studies with different asbestos fibers had shown tumor induction. The aim of this study was to identify possible carcinogenic effects of MWCNTs. We compared induced tumors with asbestos-induced mesotheliomas and evaluated their relevance for humans by immunohistochemical methods. METHODS: A total of 500 male Wistar rats (50 per group) were treated once by intraperitoneal injection with 109 or 5 × 109 WHO carbon nanotubes of one of four different MWCNTs suspended in artificial lung medium, which was also used as negative control. Amosite asbestos (108 WHO fibers) served as positive control. Morbid rats were sacrificed and necropsy comprising all organs was performed. Histopathological classification of tumors and, additionally, immunohistochemistry were conducted for podoplanin, pan-cytokeratin, and vimentin to compare induced tumors with malignant mesotheliomas occurring in humans. RESULTS: Treatments induced tumors in all dose groups, but incidences and times to tumor differed between groups. Most tumors were histologically and immunohistochemically classified as malignant mesotheliomas, revealing a predominantly superficial spread on the serosal surface of the abdominal cavity. Furthermore, most tumors showed invasion of peritoneal organs, especially the diaphragm. All tested MWCNT types caused mesotheliomas. We observed highest frequencies and earliest appearances after treatment with the rather straight MWCNT types A and B. In the MWCNT C groups, first appearances of morbid mesothelioma-bearing rats were only slightly later. Later during the two-year study, we found mesotheliomas also in rats treated with MWCNT D - the most curved type of nanotubes. Malignant mesotheliomas induced by intraperitoneal injection of different MWCNTs and of asbestos were histopathologically and immunohistochemically similar, also compared with mesotheliomas in man, suggesting similar pathogenesis. CONCLUSION: We showed a carcinogenic effect for all tested MWCNTs. Besides aspect ratio, curvature seems to be an important parameter influencing the carcinogenicity of MWCNTs.

Nanoparticles and pop-off technique for electron microscopy: a known technique for a new purpose.

Because of the size of the nanoparticles, their detection and exact anatomical localization in tissue samples are very difficult. Consequently, suitable methods are needed to prove their presence, especially co-localized to histological lesions. Therefore, the aim of this study was to investigate whether nanoparticles were detectable in specimens after reprocessing samples from glass slides using the pop-off technique. Tissue slides containing agglomerates of titanium dioxide nanoparticles already visible on a light microscopic level and amorphous silicium dioxide (SiO2) particles not observable in tissue slides were reprocessed. Furthermore, cytospots of bronchoalveolar lavage acquired from rats that previously inhaled carbon nanotubes were used. After reprocessing the samples, they were investigated using transmission electron microscopy. In all the reprocessed samples, the respective nanoparticles were detectable. Even the light microscopically invisible amorphous SiO2 particles were observed as electron dense structures. Titanium and silicium were additionally confirmed in the respective nanoparticles by energy-dispersive X-ray spectroscopy (EDX). In summary, the pop-off technique represents a fast and easy way to detect nanoparticles in histological sections. This enables further characterization of these particles by additional techniques such as EDX, and their direct correlation with light microscopic lesions at exactly the same location is investigated.

Cell counting in human endobronchial biopsies--disagreement of 2D versus 3D morphometry.

QUESTION: Inflammatory cell numbers are important endpoints in clinical studies relying on endobronchial biopsies. Assumption-based bidimensional (2D) counting methods are widely used, although theoretically design-based stereologic three-dimensional (3D) methods alone offer an unbiased quantitative tool. We assessed the method agreement between 2D and 3D counting designs in practice when applied to identical samples in parallel. MATERIALS AND METHODS: Biopsies from segmental bronchi were collected from healthy non-smokers (n = 7) and smokers (n = 7), embedded and sectioned exhaustively. Systematic uniform random samples were immunohistochemically stained for macrophages (CD68) and T-lymphocytes (CD3), respectively. In identical fields of view, cell numbers per volume unit (NV) were assessed using the physical disector (3D), and profiles per area unit (NA) were counted (2D). For CD68+ cells, profiles with and without nucleus were separately recorded. In order to enable a direct comparison of the two methods, the zero-dimensional CD68+/CD3+-ratio was calculated for each approach. Method agreement was tested by Bland-Altmann analysis. RESULTS: In both groups, mean CD68+/CD3+ ratios for NV and NA were significantly different (non-smokers: 0.39 and 0.68, p<0.05; smokers: 0.49 and 1.68, p<0.05). When counting only nucleated CD68+ profiles, mean ratios obtained by 2D and 3D counting were similar, but the regression-based Bland-Altmann analysis indicated a bias of the 2D ratios proportional to their magnitude. This magnitude dependent deviation differed between the two groups. CONCLUSIONS: 2D counts of cell and nuclear profiles introduce a variable size-dependent bias throughout the measurement range. Because the deviation between the 3D and 2D data was different in the two groups, it precludes establishing a 'universal conversion formula'.

Proliferative and non-proliferative lesions of the rat and mouse soft tissue, skeletal muscle and mesothelium.

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying lesions observed in the soft tissues including skeletal muscle as well as the mesothelium of rats and mice. The standardized nomenclature of lesions presented in this document is also available electronically on the Internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous developmental and aging lesions as well as those induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for lesions in soft tissues, skeletal muscle and mesothelium in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists. (DOI: 10.1293/tox.26.1S; J Toxicol Pathol 2013; 26: 1S-26S).

Evaluation of immunohistochemical markers to detect the genotoxic mode of action of fine and ultrafine dusts in rat lungs.

Data on local genotoxicity after particle exposure are crucial to resolve mechanistic aspects such as the impact of chronic inflammation, types of DNA damage, and their role in lung carcinogenesis. We established immunohistochemical methods to quantify the DNA damage markers poly(ADP-ribose) (PAR), phosphorylated H2AX (γ-H2AX), 8-hydroxyguanosine (8-OH-dG), and 8-oxoguanine DNA glycosylase (OGG1) in paraffin-embedded tissue from particle-exposed rats. The study was based on lungs from a subchronic study that was part of an already published carcinogenicity study where rats had been intratracheally instilled with saline, quartz DQ12, amorphous silica (Aerosil(®) 150), or carbon black (Printex(®) 90) at monthly intervals for 3 months. Lung sections were stained immunohistochemically and markers were quantified in alveolar lining cells. Local genotoxicity was then correlated with already defined endpoints, i.e. mean inflammation score, bronchoalveolar lavage parameters, and carcinogenicity. Genotoxicity was most pronounced in quartz DQ12-treated rats, where all genotoxicity markers gave statistically significant positive results, indicating considerable genotoxic stress such as occurrence of DNA double-strand breaks (DSB), and oxidative damage with subsequent repair activity. Genotoxicity was less pronounced for Printex(®) 90, but significant increases in γ-H2AX- and 8-OH-dG-positive nuclei and OGG1-positive cytoplasm were nevertheless detected. In contrast, Aerosil(®) 150 significantly enhanced only 8-OH-dG-positive nuclei and oxidative damage-related repair activity (OGG1) in cytoplasm. In the present study, γ-H2AX was the most sensitive genotoxicity marker, differentiating best between the three types of particles. The mean number of 8-OH-dG-positive nuclei, however, correlated best with the mean inflammation score at the same time point. This methodological approach enables integration of local genotoxicity testing in subchronic inhalation studies and makes immunohistochemical detection, in particular of γ-H2AX and 8-hydroxyguanine, a very promising approach for local genotoxicity testing in lungs, with prognostic value for the long-term outcome of particle exposure.

Repeatability of and relationship between potential COPD biomarkers in bronchoalveolar lavage, bronchial biopsies, serum, and induced sputum.

Chronic Obstructive Pulmonary Disease (COPD) is a chronic inflammatory disease, primarily affecting the airways. Stable biomarkers characterizing the inflammatory phenotype of the disease, relevant for disease activity and suited to predict disease progression are needed to monitor the efficacy and safety of drug interventions. We therefore analyzed a large panel of markers in bronchoalveolar lavage, bronchial biopsies, serum and induced sputum of 23 healthy smokers and 24 smoking COPD patients (GOLD II) matched for age and gender. Sample collection was performed twice within a period of 6 weeks. Assays for over 100 different markers were validated for the respective matrices prior to analysis. In our study, we found 51 markers with a sufficient repeatability (intraclass correlation coefficient >0.6), most of these in serum. Differences between groups were observed for markers from all compartments, which extends (von-Willebrand-factor) and confirms (e.g. C-reactive-protein, interleukin-6) previous findings. No correlations between lung and serum markers were observed, including A1AT. Airway inflammation defined by sputum neutrophils showed only a moderate repeatability. This could be improved, when a combination of neutrophils and four sputum fluid phase markers was used to define the inflammatory phenotype.In summary, our study provides comprehensive information on the repeatability and interrelationship of pulmonary and systemic COPD-related markers. These results are relevant for ongoing large clinical trials and future COPD research. While serum markers can discriminate between smokers with and without COPD, they do not seem to sufficiently reflect the disease-associated inflammatory processes within the airways.