Evaluating the uptake, intracellular fate and functional consequences of hepatocyte exposure to a range of nanoparticles, in vitro

The liver is recognised as a potential target site for nanoparticle (NP) toxicity, as NPs have been observed to accumulate within this organ subsequent to exposure via injection, inhalation or instillation. The liver's unique structure has to be taken into consideration when evaluating NP toxicity, as a variety of cell types of distinct morphology and function are evident, and potentially affected by NP exposure. Of particular interest are hepatocytes, due to their abundance and importance to the maintenance of normal liver function, and macrophages due to their role in host defence. The uptake and intracellular fate of fluorescent polystyrene particles (20nm and 200nm) by hepatocytes was evaluated (with exposure times of up to 60 minutes). Within the studies conducted comparisons of the response of primary rat hepatocytes, with C3A and HepG2 hepatocyte cell lines to NP exposure were made in order to investigate whether cell lines are a relevant model of hepatocyte behaviour. It was found that the uptake of particles by the primary hepatocytes, and both cell lines was size and time dependent. Specifically, it appeared that the internalisation of 200nm particles was limited, occurred at later time points (60 minutes), with the majority of particles evident at the cell surface. Polystyrene NPs (20nm) were internalised by cells after a 10 minute exposure time, after which NPs compartmentalised either within and/or between adjacent cells. The nature of the NP ‘compartments', and therefore fate of internalised NPs was then investigated to determine if the compartments developed as a consequence of the mechanism of uptake, or due to the attempted elimination of NPs from cells. It was found that NPs were not contained within early endosomes or lysosomes. However it was apparent that polystyrene NPs were eliminated to a limited extent within the bile canaliculi of all cell types, and may accumulate within the mitochondria of cell lines after a 60 minute exposure, which warrants further investigation. The impact of the PARTICLE_RISK particle panel [consisting of ultrafine carbon black (ufCB), CB, carbon nanotubes (CNTs), C60 (carbon fullerene) QD621 (positively charged quantum dots) and QD620 (negatively charged quantum dots)] on hepatocyte function was then determined. It was consistently observed that QD621 and QD620 were able to elicit the greatest extent of toxicity, evidenced within their ability to deplete cellular GSH, induce cytotoxicity, initiate a pro-inflammatory response (indicated by an increase in IL-8 production) and decrease bile secretion, in the hepatocyte couplet, in vitro model. It was observed that the pattern of response was similar within the cell lines and primary cells. Differentiated monocytic THP-1 cells (to represent the resident liver macrophages, Kupffer cells) were exposed to the PARTICLE_RISK particle panel to obtain conditioned medium (CM) that was exposed to hepatocytes, in order to gain insight into the ability of macrophages to influence NP mediated toxicity to hepatocytes. Firstly, the response of macrophages to particle exposure was considered and it was apparent that the toxicity that was observed within hepatocytes was paralleled within the response of differentiated monocytic cells (THP-1). Accordingly, QD621 were again proven to have the greatest toxic potential, with QD620 able to induce toxicity to a more limited extent. The exposure of hepatocytes to CM potentiated the toxicity observed when cells were exposed to particles alone, so that the pattern of response was comparable, but the extent of toxicity greater, and evident at earlier time points. It was apparent that QDs were able to induce an inflammatory response (characterised by TNFα and IL-8 production) within the liver that was primarily mediated by macrophages. When considering the results from all experiments it is evident that some of the particles contained within the PARTICLE_RISK panel were more capable of eliciting toxicity within the liver, and that their toxicity can be ranked in the following order: QD621>QD620>CNT=ufCB=C60>CB.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Evaluating the uptake, intracellular fate and functional consequences of hepatocyte exposure to a range of nanoparticles, in vitro

The liver is recognised as a potential target site for nanoparticle (NP) toxicity, as NPs have been observed to accumulate within this organ subsequent to exposure via injection, inhalation or instillation. The liver’s unique structure has to be taken into consideration when evaluating NP toxicity, as a variety of cell types of distinct morphology and function are evident, and potentially affected by NP exposure. Of particular interest are hepatocytes, due to their abundance and importance to the maintenance of normal liver function, and macrophages due to their role in host defence. The uptake and intracellular fate of fluorescent polystyrene particles (20nm and 200nm) by hepatocytes was evaluated (with exposure times of up to 60 minutes). Within the studies conducted comparisons of the response of primary rat hepatocytes, with C3A and HepG2 hepatocyte cell lines to NP exposure were made in order to investigate whether cell lines are a relevant model of hepatocyte behaviour. It was found that the uptake of particles by the primary hepatocytes, and both cell lines was size and time dependent. Specifically, it appeared that the internalisation of 200nm particles was limited, occurred at later time points (60 minutes), with the majority of particles evident at the cell surface. Polystyrene NPs (20nm) were internalised by cells after a 10 minute exposure time, after which NPs compartmentalised either within and/or between adjacent cells. The nature of the NP ‘compartments’, and therefore fate of internalised NPs was then investigated to determine if the compartments developed as a consequence of the mechanism of uptake, or due to the attempted elimination of NPs from cells. It was found that NPs were not contained within early endosomes or lysosomes. However it was apparent that polystyrene NPs were eliminated to a limited extent within the bile canaliculi of all cell types, and may accumulate within the mitochondria of cell lines after a 60 minute exposure, which warrants further investigation.The impact of the PARTICLE_RISK particle panel [consisting of ultrafine carbon black (ufCB), CB, carbon nanotubes (CNTs), C60 (carbon fullerene) QD621 (positively charged quantum dots) and QD620 (negatively charged quantum dots)] on hepatocyte function was then determined. It was consistently observed that QD621 and QD620 were able to elicit the greatest extent of toxicity, evidenced within their ability to deplete cellular GSH, induce cytotoxicity, initiate a pro-inflammatory response (indicated by an increase in IL-8 production) and decrease bile secretion, in the hepatocyte couplet, in vitro model. It was observed that the pattern of response was similar within the cell lines and primary cells. Differentiated monocytic THP-1 cells (to represent the resident liver macrophages, Kupffer cells) were exposed to the PARTICLE_RISK particle panel to obtain conditioned medium (CM) that was exposed to hepatocytes, in order to gain insight into the ability of macrophages to influence NP mediated toxicity to hepatocytes. Firstly, the response of macrophages to particle exposure was considered and it was apparent that the toxicity that was observed within hepatocytes was paralleled within the response of differentiated monocytic cells (THP-1). Accordingly, QD621 were again proven to have the greatest toxic potential, with QD620 able to induce toxicity to a more limited extent. The exposure of hepatocytes to CM potentiated the toxicity observed when cells were exposed to particles alone, so that the pattern of response was comparable, but the extent of toxicity greater, and evident at earlier time points. It was apparent that QDs were able to induce an inflammatory response (characterised by TNFα and IL-8 production) within the liver that was primarily mediated by macrophages. When considering the results from all experiments it is evident that some of the particles contained within the PARTICLE_RISK panel were more capable of eliciting toxicity within the liver, and that their toxicity can be ranked in the following order: QD621>QD620>CNT=ufCB=C60>CB

Nanotoxicology

As the production and use of nanomaterials (NMs) in medicine and many other applications develops, so the need to understand the potential risks posed by NMs to human health (and the environment) increases (Aitken et al. 2006). At the nanoscale (1-100 nm), materials exhibit properties that are different to larger or bulk materials. These new properties are exploited by researchers and industry to generate new products; however, the same properties can also inuence how the NM behaves in biological systems, including affecting toxicity. Nanotoxicology is a relatively new eld of research that aims to assess the human and environmental hazard of nanomaterials. In recent years, this new discipline has seen a rapid expansion in the number of studies concerned with assessing the safety of engineered NMs (Figure 20.1)

Using 3D gastrointestinal tract in vitro models with microfold cells and mucus secreting ability to assess the hazard of copper oxide nanomaterials

Abstract: Background: Copper oxide nanomaterials (CuO NMs) are exploited in many products including inks, cosmetics, textiles, wood preservatives and food contact materials. Their incorporation into these products may enhance oral exposure in consumer, environmental and occupational settings. Undifferentiated and differentiated monocultures of Caco-2 cells are commonly used to assess NM toxicity to the intestine in vitro. However, the integration of other cell types into Caco-2 in vitro models increases their physiological relevance. Therefore, the aim of this study is to evaluate the toxicity of CuO NMs and copper sulphate ( CuSO4) to intestinal microfold (M) cell (Caco-2/Raji B) and mucus secreting (Caco-2/HT29-MTX) co-culture in vitro models via assessment of their impact on barrier integrity, viability and interleukin (IL)-8 secretion. The translocation of CuO NMs and CuSO4 across the intestinal barrier was also investigated in vitro. Results: CuO NMs and CuSO4 impaired the function of the intestinal barrier in the co-culture models [as indicated by a reduction in transepithelial electrical resistance (TEER) and Zonular occludens (ZO-1) staining intensity]. Cu translocation was observed in both models but was greatest in the Caco-2/Raji B co-culture. CuO NMs and CuSO4 stimulated an increase in IL-8 secretion, which was greatest in the Caco-2/HT29-MTX co-culture model. CuO NMs and CuSO4 did not stimulate a loss of cell viability, when assessed using light microscopy, nuclei counts and scanning electron microscopy. CuO NMs demonstrated a relatively similar level of toxicity to CuO4 in both Caco-2/Raji B and Caco-2/ HT29-MTX co- culture models. Conclusions: The Caco-2/Raji B co-culture model was more sensitive to CuO NM and CuSO4 toxicity than the Caco-2/HT29-MTX co-culture model. However, both co-culture models were less sensitive to CuO NM and CuSO4 toxicity than simple monocultures of undifferentiated and differentiated Caco-2 cells, which are more routinely used to investigate NM toxicity to the intestine. Obtained data can therefore feed into the design of future studies which assess the toxicity of substances (e.g. NMs) and pathogens to the intestine (e.g. by informing model and endpoint selection). However, more testing with a wider panel of NMs would be beneficial in order to help select which in vitro models and endpoints to prioritise when screening the safety of ingested NMs. Comparisons with in vivo findings will also be essential to identify the most suitable in vitro model to screen the safety of ingested NMs

In Utero Exposure to Particulate Air Pollution during Pregnancy:Impact on Birth Weight and Health through the Life Course

In high-income countries, and increasingly in lower-and middle-income countries, chronic non-communicable diseases (NCDs) have become the primary health burden. It is possible that in utero exposure to environmental pollutants such as particulate matter (PM) may have an impact on health later in life, including the development of NCDs. Due to a lack of data on foetal growth, birth weight is often used in epidemiologic studies as a proxy to assess impacts on foetal development and adverse birth outcomes since it is commonly recorded at birth. There are no research studies with humans that directly link PM exposure in utero to birth weight (BW) and subsequently, the effects of lower BW on health outcomes in old age. It is, however, plausible that such associations exist, and it is thus important to assess the potential public health impacts of PM across the life course, and it is plausible to use birth weight as an indicator of risk. We therefore split this narrative review into two parts. In the first part, we evaluated the strength of the evidence on the impact of PM exposure during the entire pregnancy on birth weight outcomes in ten meta-analyses. In the second part, we reviewed the literature linking lower birth weight to childhood and adult chronic cardiovascular disease to explore the potential implications of PM exposure in utero on health later in life. Within the reviewed meta-studies on birth weight, there is sufficient evidence that PM pollution is associated with lower birth weight, i.e., the majority of meta-studies found statistically significant reductions in birth weight. From the second part of the review, it is evident that there is good evidence of associations between lower birth weight and subsequent cardiovascular disease risk. It is thus plausible that in utero exposure to PM is associated with lower birth weight and persisting biological changes that could be associated with adverse health effects in adulthood. Based on the reviewed evidence, however, the magnitude of later life cardiovascular health impacts from in utero exposure and its impact on BW are likely to be small compared to health effects from exposure to particulate air pollution over a whole lifetime.</p

Identification of the mechanisms that drive the toxicity of TiO2 particulates: the contribution of physicochemical characteristics

This review focuses on outlining the toxicity of titanium dioxide (TiO&#x2082;) particulates in vitro and in vivo, in order to understand their ability to detrimentally impact on human health. Evaluating the hazards associated with TiO&#x2082; particles is vital as it enables risk assessments to be conducted, by combining this information with knowledge on the likely exposure levels of humans. This review has concentrated on the toxicity of TiO&#x2082;, due to the fact that the greatest number of studies by far have evaluated the toxicity of TiO&#x2082;, in comparison to other metal oxide particulates. This derives from historical reasons (whereby the size dependency of particulate toxicity was first realised for TiO&#x2082;) and due to its widespread application within consumer products (such as sunscreens). The pulmonary and dermal hazards of TiO&#x2082; have been a particular focus of the available studies, due to the past use of TiO&#x2082; as a (negative) control when assessing the pulmonary toxicity of particulates, and due to its incorporation within consumer products such as sunscreens. Mechanistic processes that are critical to TiO&#x2082; particulate toxicity will also be discussed and it is apparent that, in the main, the oxidant driven inflammatory, genotoxic and cytotoxic consequences associated with TiO&#x2082; exposure, are inherently linked, and are evident both in vivo and in vitro. The attributes of TiO&#x2082; that have been identified as being most likely to drive the observed toxicity include particle size (and therefore surface area), crystallinity (and photocatalytic activity), surface chemistry, and particle aggregation/agglomeration tendency. The experimental set up also influences toxicological outcomes, so that the species (or model) used, route of exposure, experiment duration, particle concentration and light conditions are all able to influence the findings of investigations. In addition, the applicability of the observed findings for particular TiO&#x2082; forms, to TiO&#x2082; particulates in general, requires consideration. At this time it is inappropriate to consider the findings for one TiO&#x2082; form as being representative for TiO&#x2082; particulates as a whole, due to the vast number of available TiO&#x2082; particulate forms and large variety of potential tissue and cell targets that may be affected by exposure. Thus emphasising that the physicochemical characteristics are fundamental to their toxicity