An In Vitro Versus In Vivo Toxicogenomic Investigation of Prenatal Exposures to Tobacco Smoke.

Approximately 1 million women smoke during pregnancy despite evidence demonstrating serious juvenile and/or adult diseases being linked to early-life exposure to cigarette smoke. Susceptibility could be determined by factors in previous generations, that is, prenatal or "maternal" exposures to toxins. Prenatal exposure to airborne pollutants such as mainstream cigarette smoke has been shown to induce early-life insults (i.e., gene changes) in Offspring that serve as biomarkers for disease later in life. In this investigation, we have evaluated genome-wide changes in the lungs of mouse Dams and their juvenile Offspring exposed prenatally to mainstream cigarette smoke. An additional lung model was tested alongside the murine model, as a means to find an alternative in vitro, human tissue-based replacement for the use of animals in medical research. Our toxicogenomic and bio-informatic results indicated that in utero exposure altered the genetic patterns of the fetus, which could put them at greater risk for developing a range of chronic illnesses in later life. The genes altered in the in vitro, cell culture model were reflected in the murine model of prenatal exposure to mainstream cigarette smoke. The use of alternative in vitro models derived from human medical waste tissues could be viable options to achieve human endpoint data and conduct research that meets the remits for scientists to undertake the 3Rs practices.

Particulate matter from both heavy fuel oil and diesel fuel shipping emissions show strong biological effects on human lung cells at realistic and comparable in vitro exposure conditions.

BACKGROUND: Ship engine emissions are important with regard to lung and cardiovascular diseases especially in coastal regions worldwide. Known cellular responses to combustion particles include oxidative stress and inflammatory signalling. OBJECTIVES: To provide a molecular link between the chemical and physical characteristics of ship emission particles and the cellular responses they elicit and to identify potentially harmful fractions in shipping emission aerosols. METHODS: Through an air-liquid interface exposure system, we exposed human lung cells under realistic in vitro conditions to exhaust fumes from a ship engine running on either common heavy fuel oil (HFO) or cleaner-burning diesel fuel (DF). Advanced chemical analyses of the exhaust aerosols were combined with transcriptional, proteomic and metabolomic profiling including isotope labelling methods to characterise the lung cell responses. RESULTS: The HFO emissions contained high concentrations of toxic compounds such as metals and polycyclic aromatic hydrocarbon, and were higher in particle mass. These compounds were lower in DF emissions, which in turn had higher concentrations of elemental carbon ("soot"). Common cellular reactions included cellular stress responses and endocytosis. Reactions to HFO emissions were dominated by oxidative stress and inflammatory responses, whereas DF emissions induced generally a broader biological response than HFO emissions and affected essential cellular pathways such as energy metabolism, protein synthesis, and chromatin modification. CONCLUSIONS: Despite a lower content of known toxic compounds, combustion particles from the clean shipping fuel DF influenced several essential pathways of lung cell metabolism more strongly than particles from the unrefined fuel HFO. This might be attributable to a higher soot content in DF. Thus the role of diesel soot, which is a known carcinogen in acute air pollution-induced health effects should be further investigated. For the use of HFO and DF we recommend a reduction of carbonaceous soot in the ship emissions by implementation of filtration devices.

A normal and biotransforming model of the human bronchial epithelium for the toxicity testing of aerosols and solubilised substances.

In this article, we provide an overview of the experimental workflow by the Lung and Particle Research Group at Cardiff University, that led to the development of the two in vitro lung models - the normal human bronchial epithelium (NHBE) model and the lung-liver model, Metabo-Lung™. This work was jointly awarded the 2013 Lush Science Prize. The NHBE model is a three-dimensional, in vitro, human tissue-based model of the normal human bronchial epithelium, and Metabo-Lung involves the co-culture of the NHBE model with primary human hepatocytes, thus permitting the biotransformation of inhaled toxicants in an in vivo-like manner. Both models can be used as alternative test systems that could replace the use of animals in research and development for safety and toxicity testing in a variety of industries (e.g. the pharmaceutical, environmental, cosmetics, and food industries). Metabo-Lung itself is a unique tool for the in vitro detection of toxins produced by reactive metabolites. This 21st century animal replacement model could yield representative in vitro predictions for in vivo toxicity. This advancement in in vitro toxicology relies on filter-well technology that will enable a wide-spectrum of researchers to create viable and economic alternatives for respiratory safety assessment and disease-focused research.

Tissue-Specific stem cell differentiation in an in vitro airway model.

The respiratory tract is the primary site of exposure to airborne compounds, with the bronchial epithelium providing one of the first lines of defence. A growing need exists for an accurate in vitro model of the bronchial epithelium. Here, normal human bronchial epithelial (NHBE) cells cultured at an air/liquid interface create a fully differentiated, in-vivo-like model of the human bronchial epithelium. Developmental characterisation includes (i) trans-epithelial electrical resistance, (ii) morphology and (iii) bronchial cell specific stains/markers. It is concluded that the basal/progenitor cells create a pseudo-stratified, mucociliary NHBE model containing basal, serous, Clara, goblet and ciliated cells, reflective of the normal human bronchial epithelium (days 24-33 ALI culture).

Human lung tissue engineering: a critical tool for safer medicines.

In the field of human tissue-engineering, there has been a strong focus on the clinical aspects of the technology, i.e. repair, replace and enhance a given tissue/organ. However, much wider applications for tissue engineering (TE) exist outside of the clinic that are often not recognised, and include engineering more relevant models than animals in basic research and safety testing. Traditionally, research is initially conducted on animals or cell lines, both of which have their limitations. With regard to cell lines, they are usually transformed to enable indefinite proliferation. These immortalised cell lines provide the researcher with an almost limitless source of material. However, the pertinence of the data produced is now under scrutiny, with the suggestion that some historical cell lines may not be the cell type originally reported. By engineering normal, biomimetic (i.e. life-mimicking), human tissues with defined physiology (i.e. human tissue equivalents), the complex 3-dimensional (3-D) tissue/organ physiology is captured in vitro, providing the opportunity to directly replace the use of animals in research/testing with more relevant systems. Therefore, it is imperative that testing strategies using organotypic models are developed that can address the limitations of current animal and cellular models and thus improve drug development, enabling faster delivery of drugs which are safer, more effective and have fewer side effects in humans.

Human primary bronchial lung cell constructs: the new respiratory models.

Scientists routinely work within the three R's principles of 'Reduction, Refinement and Replacement' of animal experiments. Accordingly, viable alternatives are regularly developed, and in the specific case of the human lung, in vitro models for inhalation toxicology that mimic in vivo toxic events that may occur in the human lung, are welcomed. This is especially warranted given the new EU regulations (i.e. REACH) coming into force for the handling of chemicals and the advent of nanotoxicology. Furthermore, recent advances in human tissue-engineering has made it feasible and cost effective to construct human tissue equivalents of the respiratory epithelia, as in-house models derived from primary cells. There is an urgent need for engineered tissue equivalents of the lung given the increase in pharmaceutically valuable drugs, toxicity testing of environmental pollutants and the advent of nanotoxicology. Given the well-known problems with 2-dimensional (2-D) cell cultures as test beds, more realistic 3-D tissue constructs are required, especially for preclinical stages of cell- and tissue-based, high-throughput screening in drug discovery. The generation of high-fidelity engineered tissue constructs is based on the targeted interactions of organ-specific cells and intelligent biomimetic scaffolds which emulate the natural environment of their native extracellular matrix, in which the cells develop, differentiate and function. The proximal region of the human respiratory system is a critical zone to recapitulate for use as in vitro alternatives to in vivo inhalation toxicology. Undifferentiated normal human bronchial epithelia cells can be obtained from surgical procedures or purchased from commercial sources and used to establish 3-D, differentiated, organo-typic cell cultures for pulmonary research.

Filter-well technology for advanced three-dimensional cell culture: perspectives for respiratory research.

Cell culture has long been a valuable tool for studying cell behaviour. Classical plastic substrates are two-dimensional, and usually promote cellular proliferation and inhibit differentiation. Understanding cell behaviour within complex multicellular tissues requires the systematic study of cells within the context of specific model microenvironments. A model system must mimic, to a certain degree, the in vivo situation, but, at the same time, can significantly reduce its complexity. There is increasing agreement that moving up to the third dimension provides a more physiologically-relevant and predictive model system. Moreover, many cellular processes (morphogenesis, organogenesis and pathogenesis) have been confirmed to occur exclusively when cells are ordered in a three-dimensional (3-D) manner. In order to achieve the desired in vivo phenotype, researchers can use microporous membranes for improved in vitro cell culture experiments. In the present review, we discuss the applications of filter-well technology for the advanced 3-D cell culture of human pulmonary cells.