17.5 Animal Studies ...30917.5.1 Bronchoalveolar Lavage...309 17.5.2 Intratracheal Instillation ...309 17.5.3 Inhalation Studies ...309 17.5.4 Monitoring Exposure, Dose, and Response ...
Trang 117 Approaches to the Toxicological
Testing of Particles
Ken Donaldson
MRC/University of Edinburgh Centre for Inflammation Research,
Queen’s Medical Research Institute
Steve Faux
MRC/University of Edinburgh Centre for Inflammation Research,
Queen’s Medical Research Institute
Paul J A Borm
Centre of Expertise in Life Sciences (CEL), Hogeschool Zuyd
Vicki Stone
School of Life Sciences, Napier University
CONTENTS
17.1 Background to Testing of Particles 300
17.2 Factors Affecting Particle Toxicity 300
17.2.1 Particle Size 300
17.2.1.1 Particle Shape 301
17.2.1.2 Particle-Derived Transition Metals 302
17.2.1.3 Particle-Derived Free Radicals 303
17.2.1.4 Particle Biopersistence 303
17.3 Approaches to Testing 304
17.3.1 Characterizing the Particles 304
17.4 Assessment of Toxicity In Vitro 304
17.4.1 Cytotoxicity 305
17.4.2 Cell Stimulation 305
17.4.3 Cell Proliferation 305
17.4.4 Cytokine Measurement 306
17.4.5 Oxidative Stress Measurement 306
17.4.6 Alterations in Cell Signaling Cascades 306
17.4.6.1 Intracellular Calcium 306
17.4.6.2 Mitogen-Activated Protein Kinase Cascade 307
17.4.6.3 Transcription Factor Activation 307
17.4.7 Effects on Blood 308
17.4.7.1 Endothelium 308
17.4.7.2 Platelets 308
17.4.7.3 Atherosclerotic Plaques 308
17.4.8 Effects on the Brain 308
17.4.9 In Vitro Tests for Genotoxicity 309
299
Trang 217.5 Animal Studies 309
17.5.1 Bronchoalveolar Lavage 309
17.5.2 Intratracheal Instillation 309
17.5.3 Inhalation Studies 309
17.5.4 Monitoring Exposure, Dose, and Response 310
17.5.5 Immunological Effects of Particles 310
17.5.6 Effects on the Microbicidal Activity of the Lungs 310
17.5.7 Animal Models to Study the Cardiovascular Effects of Particles 311
17.5.8 Animal Models of Neurological Effects of Particles 311
17.5.9 Conclusion—A Tiered Approach to Testing of a New Particle 311
References 312
17.1 BACKGROUND TO TESTING OF PARTICLES
There is a wide spectrum of different approaches to the testing of particles that range from long-term inhalation studies, with the final endpoint of cancer, to short-long-term tests in vitro delong-termining the ability of the particle to modulate cellular functions The value of each of these tests in predicting pathogenicity varies; but in general, there is a playoff between the extended timescale and high cost
of long-term pathogenicity experiments in animals which can be used in risk assessment and the short timescale and relative inexpensiveness of in vitro data, which can, at best, be used in hazard assessment If epidemiological or clinical data were available on the pathogenic outcome of exposure to a novel particle, then this would form the basis of a rational test strategy However,
in the absence of such information, a strategy based on knowledge of structure, chemistry, and shape of the particle could allow benchmarking to similar particles of known pathogenicity in order
to decide on the most appropriate endpoint
This spectrum of possible pathology arising from particle exposure poses an immediate problem for a testing strategy A strategy designed to detect a carcinogenic endpoint, for example, would be entirely different from one that would be chosen to detect the potential to cause asthma So there is a need for some knowledge of the likely pathology that would arise This could be provided by:
1 A priori knowledge from clinical observations or epidemiological studies indicating that
a particular disease or manifestation of toxicity is associated with exposure to the particle
2 In the absence of such information, there could be benchmarking to particles of known toxicity For example, if the particle type was a mineral and contained some quartz, then the endpoints of fibrosis and cancer could be selected If the particle was organic or contained heavy metals, then sensitization might be considered Fibrous particles would
be suspected of causing mesothelioma, etc
17.2 FACTORS AFFECTING PARTICLE TOXICITY
Given the different diseases caused by particles, we assume that there are different target cells and tissues (e.g., airways, alveolar cells, immune cells) that are affected by different particles This can
be understood from the point of view of differences in dose to these different target cells caused by
Trang 3differences in the distribution of dose throughout the respiratory tract because of variation in particle size
The fractional deposition of various sized particles for different pulmonary compartments is dictated by the aerodynamic diameter, Dae, as shown Table 17.1 This is defined as the diameter of a particle of unit density with the same falling speed as the particle of interest
Different-sized particles would be expected to elicit different types of pathogenic response because they deposit in different regions, as shown in Table 17.2
From the foregoing, it can be concluded that the characterization of size of a novel particle is important in deciding what is the most likely endpoint to examine, since size will bear directly on the site of deposition and the subsequent response When the site of the adverse health effect is not the local lung environment, it is more difficult to know which site of deposition is most important because there could be, in theory at least, translocation from any site of deposition—hence the question marks over the site of deposition of particles that are associated with mesothelioma and strokes/heart attacks
In the case of nanoparticles (see later) additional targets, such as the blood and brain may be relevant, as a consequence of translocation
17.2.1.1 Particle Shape
Particles come in a number of different shapes, e.g., compact, platy, and fibrous The importance of particle shape is best understood for fibers, and fiber length is known to be a major factor in pathogenicity as reviewed in Donaldson and Tran (2004) Long fibers of amosite asbestos were much more pathogenic than a sample of the same material milled so that the fiber length was drastically shortened, with much of the sample being so short that it was classified as non-fibrous (Davis et al 1986) For other particles, shape is less obvious as a parameter that mediates toxicity
TABLE 17.2
The Adverse Effects Resulting from Different Sizes of Particles
Depositing in Different Compartments
Deposition Site Disease
Thoracic COPD, asthma, central lung cancer
Respirable Small airways disease, peripheral lung cancer, emphysema,
interstitial fibrosis, cardiovascular effects
TABLE 17.1
Anatomical Site of Deposition for the Different Deposition Fractions
Deposition Fraction Approx AerodynamicDiameter (mm) Definition Site of Deposition Inhalable w50–100 The fraction inhaled through
the nose and mouth Mouth, larynx, pharynx Thoracic w10–50 Fraction penetrating beyond
the larynx
The above plus airways Respirable !w10 Fraction penetrating beyond
the ciliated airways
The above plus terminal bronchioles and alveolar ducts
Trang 4Shape may not be the only factor that dictates fiber pathogenicity—even amongst long and thin fibers there are differences in pathogenicity, especially in mesothelioma production Erionite (Maltoni, Minardi, and Morisi 1982) and silicon carbide fibers (Davis et al 1996), for example were much more active in causing mesothelioma following inhalation exposure than would be expected from their dimensions For this reason it is likely that another factor, surface reactivity (see below), could be important in mediating some of their pathogenicity
17.2.1.2 Particle-Derived Transition Metals
It has become evident that transition metals are key players in the pro-inflammatory effects of a range of particle types (Ghio et al 2006) Iron is especially important because it has the ability to generate free radicals via Fenton chemistry that is well characterized The state of the iron is all important; but in particular, the amount of Fe(II) is central since this is the directly harmful species (Ghio and Cohen 2005) Consequently, total iron is not necessarily informative as to the biologi-cally active iron The iron must redox-cycle to be capable of causing major injury to macromolecules This is accomplished by a reluctant in the region of the particle, e.g., glutathione, ascorbate, NADH, or even superoxide anion This means that the presence of anti-oxidants in the lungs is a “double-edged sword.”
By the sequence of events shown in Figure 17.1, the highly toxic and reactive hydroxyl radical can be formed The hydroxyl radical may be involved in diffusion-limited reactions which lead to the formation of various carbon-centered radicals, peroxyl, alkoxyl, and thiyl radicals, all of which have harmful consequences for cells Because different reductants could have different potencies in causing reduction of Fe(III), and because of the known role of chelating agents, the micro-environ-ment of the lung where the particle is present could be all-important in determining how much reactive iron is present at the surface In addition, the particle may accumulate biological iron, which can also have free radical-generating activity (Ghio, Jaskot, and Hatch 1994) In various models, the biological effects of several different types of ambient particle, including PM10and ROFA, have been suggested to be driven by their transition metal content (Dreher et al 1997; Jimenez et al 2000; Rice et al 2001; Dai, Xie, and Churg 2002; Molinelli et al 2002; McNeilly
et al in press) The measurement of the sum potential for a particle to generate oxidative stress has been advanced as a key parameter that might dictate overall toxicity
Particle Transition metal Reducing
environment of the lungs
Altered redox status
Inflammation
Direct tissue damage
Redox cycling
O2 and OH −
FIGURE 17.1 Generation of oxidative stress by transition metals
Trang 517.2.1.3 Particle-Derived Free Radicals
Quartz is one of the most toxic particles and it is known to have a highly reactive surface The quartz surface can generate reactive oxygen species (ROS) in several ways following interactions
of the quartz particles with pulmonary cells or lung fluids (Castranova, Dalal, and Vallyathan 1995) PM10 has been demonstrated to generate free radicals and cause oxidative stress (Ghio
et al 1996; Gilmour et al 1996; Squadrito et al 2001; Aust et al 2002) via transition metals and organics that redox cycle Nanoparticles are also capable of generating ROS in cell-free systems (Wilson et al 2002) In fact, almost all pathogenic particles studied, including asbestos (Lund and Aust 1991), glass fibers (Gilmour et al 1997), and coalmine dust (Dalal et al 1995) are capable of generating ROS in cell free systems Oxidative stress has been suggested as a generic mechanism for the action of particles (Donaldson, Beswick, and Gilmour 1996), and more recently for nanoparticles (Donaldson et al 2005; Oberdo¨rster, Oberdo¨rster, and Oberdo¨rster 2005) Many systems are available for measuring the fee-radical-generating potential of a particle sample, including purely chemical methods such as HPLC (Brown, Fisher, and Donaldson 1998), the use
of super-coiled plasmid DNA as a sensor of free radicals (Gilmour et al 1997), the use of EPR and spin traps to capture these short-lived moieties (Shi et al 2003), and calf thymus DNA to detect 8-hydroxydeoxyguanosine (8-OHdG)
17.2.1.4 Particle Biopersistence
Biopersistence is the capacity of particles to persist in the lungs Biopersistence is limited by the potential of particles to dissolve or lose elements, break, or be mechanically cleared from the lungs
by macrophages The potential for a particle to dissolve in the lungs would seem intuitively to be an important factor, since the dose of particle would not be expected to build up in the case of soluble particles However, little is know about the biochemical conditions that pertain in the lungs—the lung is largely a “black box” in this regard, although differences in pH and the impact of coating of the particles is to be anticipated
The best case where this property is seen as being important is with fibers Long fibers are not well cleared from the respiratory region of the lungs (Coin, Roggli, and Brody 1994; Searl et al 1999), presumably because of the difficulties of the alveolar macrophage to successfully phagocy-tose and then move with them to the mucociliary escalator Thus the ability of long fibers to persist
in the lungs without being either dissolved away or weakened so that they break into smaller fibers which can be easily cleared is seen as an important factor contributing to pathogenicity (Hesterberg
et al 1994) For any particle, its ability to biopersist will be an important factor in modifying its pathogenicity
TABLE 17.3 Important Particle Characteristics to Be Ascertained in Samples of Particles of Unknown Toxicity
Physical Characteristics Chemical Characteristics Dimensions Transition metals
Surface area/unit mass Quartz or cristobalite content Biopersistence Heavy metals
Free radical activity PAH Durability Endotoxin
Trang 617.3 APPROACHES TO TESTING
An understanding of the nature of the test particle with regard to shape, size, elemental compo-sition, transition metal content, endotoxin contamination, etc., is vital to the testing strategy since it will allow the particle to be benchmarked There are a number of parameters that could be assessed Once again, the concept of benchmarking is a useful one and the source of the particle, e.g., mineral-derived, man-made fiber, ash, etc., can be used to decide which is the most likely parameter that should be determined.Table 17.3shows some particle characteristics that can be quantified, which might shed light on its likely pathogenicity
Endotoxin is a potential confounder in all studies with particles and its presence should be rigor-ously monitored since it may explain all the toxicity of a dust sample (Brown and Donaldson 1996) Endotoxin can be measured by specific ELISA or functionally using the amoebocyte lysate assay
17.4 ASSESSMENT OF TOXICITY IN VITRO
The European Centre for the Validation of Alternative Methods (ECVAM) published a report on
“Nonanimal tests for evaluating the toxicity of solid xenobiotics,” and this contains recommen-dations for in vitro tests with particles (Fubini et al 1998) The majority of in vitro tests for detecting particle toxicity are aimed at detecting either direct or indirect pro-inflammatory effects Acute and chronic inflammation is thought to be central to the etiology of many lung disorders, such
as asthma and chronic obstructive pulmonary disease (COPD) The specific characteristics of the inflammatory response may be different, but all are characterized by the recruitment of inflammatory cells into the lung These activated cells, such as alveolar macrophages and neutrophils, produce cytokines and ROS and many other mediators involved in inflammation Once triggered, the inflam-matory response will persist in these conditions leading to lung injury The intracellular mechanism
in the lung epithelium and the macrophages leading to lung injury in response to environmental particulates will involve the activation and upregulation of transcription factors, such as activator protein-1 (AP-1) and nuclear factor-kB (NF-kB), leading to increased gene expression and the biosynthesis of proinflammatory mediators Particles may stimulate inflammation by a number of pathways such as:
1 Cell death, a potent stimulus for inflammation
2 Nonspecific stimulation of cell receptors
3 Oxidative stress
4 Calcium flux
5 Via the immune system (see Figure 17.2)
Particles
Cell necrosis Calcium flux
Oxidative stress
Non-specific stimulation of receptors Pro-inflammatorygene expression Inflammation
Immune system Hypersensitivity
FIGURE 17.2 Pathways for particles to cause inflammation There can be direct stimulation of target cells for pro-inflammatory gene expression, or inflammation can arise by more complex indirect routes that involve cytotoxicity or the immune system
Trang 7In vitro models are useful tools at two stages, namely the assessment of toxicity as the second tier of the testing system and the elucidation of the mechanism(s) of action A reduction in the use of experimental animals is a clear advantage of such studies, but in vitro systems also provide a
“simplified” model in which the details of the mechanism of action may be more readily examined The potential toxicity of particles is tested in vitro using either primary cells or cell lines in culture There are a number of cell types that are of obvious interest when investigating the potential effects of particles, including type I and type II epithelial cells, Clara cells, alveolar macrophages, and neutrophils Primary cells are obviously an advantage in that they are not transformed, hence they will correspond more closely to the cells found in vivo Primary cells, however, often have a limited life span in culture, and for this reason, cell lines such as the A549 human type II cell line and THP-1 monocytes are widely used, due to their ready availability and the fact that the pathways under study are similar in these permanent cell lines to freshly derived cells of the same type The acquisition of human primary cells remains difficult for many researchers, and for this reason primary rat cells are frequently used as an alternative The use
of cells has both benefits and drawbacks, both of which are well-known The benefits include the ability to dissect out the sub-cellular pathways and responses and to isolate the responses specific
to the cell type in question The drawbacks are that there is no influence of the other cell types and the circulation that ordinarily plays an important role in the responses of any single cell type
A number of reliable and well-documented techniques are available to assess viability following particle exposure, including the MTT assay, which measures the metabolic competence of cells by assessing the activity of succinate dehydrogenase enzyme activity, a key enzyme in cellular respir-ation Assessment of lactate dehydrogenase (LDH) enzyme leakage from the cells measures plasma membrane integrity The MTT assay and measurement of LDH leakage are appropriate for death via necrosis A number of particle types have been proposed to induce programmed cell death or apoptosis (Be´ruBe´ et al 1996; Iyer and Holian 1997) The techniques available for the detection of apoptosis are numerous, from detection of DNA fragmentation to commercially available cell death ELISA kits and fluorescent dyes that label the DNA, such as propidium iodide and Hoechst 33342
Many particles are thought to induce effects on the lung by mechanisms other than toxicity Some particles may in fact cause the stimulation of various cell types by, e.g., increasing entry of calcium
or stimulating kinase activity, leading to cell proliferation or an increase in the production of cytokines and other pro-inflammatory mediators Measurement of the production of ROS and cytokines, such as TNF-a and IL-1b by target cells, may form part of a testing strategy to discrimi-nate between non-pathogenic and pathogenic particulates by the ability of various particulate preparations to differentially produce these mediators There are a number of assays that can be used to assess cell stimulation in vitro and these are outlined below
Increased cell proliferation has been noted on exposure of various cell types to different particles For example, treatment of primary rat type II epithelial cells with silica for 24 h has been shown to induce cell proliferation, as assessed by the incorporation of tritiated thymidine into the DNA of dividing cells This simple technique has the disadvantage of using radioactivity, although at a low level A similar technique involves the incorporation of 5-bromo-20-deoxyuridine (BrdU) into DNA which is then assessed by immunostaining (Timblin, Janssen, and Mossman 1995) This non-radioactive technique has the advantage that BrdU can be used for observation by microscopy as well as quantification through either spectroscopy or fluorimetry
Trang 817.4.4 CYTOKINEMEASUREMENT
One of the most obvious ways to assess the potential inflammogenic activity of a particle is by measuring the output of cytokines Secreted cytokines are frequently assayed in the culture media through the use of ELISA In addition, the quantification of specific mRNA sequences, through either Northern Blotting or RT-PCR, allows further investigation of gene regulation on exposure to particles The same techniques are also applicable to other pro-inflammatory mediators
There is abundant data to suggest that many particle types induce their effects on the lungs in part through ROS, leading to oxidative stress (Tao, Gonzalez-Flecha, and Kobzik 2003) The free radicals produced at the surface of a variety of particle types along with the ROS released by leukocytes during phagocytosis and inflammation induce an oxidative stress within the lung leading
to a range of events from oxidative damage to bio-molecules, such as DNA and protein, to activation of oxidative stress-responsive transcription factors that lead to transcription of pro-inflammatory genes (Rahman and MacNee 2000; Gilmour et al 2003; Brown et al 2004a) The measurement of intracellular glutathione in its reduced (GSH) and oxidized (GSSG) forms remains
a sensitive means by which the induction of oxidative stress can be assessed GSH is one of the major intracellular antioxidants (reviewed in Karin 1998) In acting as an antioxidant, two molecules of GSH are oxidized to form GSSG, which is then reduced back to GSH by the enzyme glutathione reductase using NADPH as a source of reductant When the cell is exposed
to high levels of oxidants, NADPH within the cell is decreased, allowing depletion of GSH and an increase in GSSG The depletion of GSH is often used as a marker of oxidative stress in response to particles Enzyme assays exist to measure the activity of enzymes such as g-glutamyl transpepti-dase (g-GT), the enzyme responsible for the uptake of the components of GSH across the plasma membrane, and g-glutamylcysteine synthetase (g-GCS), the rate-limiting enzyme in the synthesis
of GSH
17.4.6.1 Intracellular Calcium
Alterations in intracellular calcium homeostasis have been implicated following oxidative stress In the resting nonstimulated cell, a Ca2CATPase pump in the plasma membrane actively extrudes
Ca2Cfrom the cell while a different Ca2CATPase pump in the endoplasmic reticulum (ER) actively sequesters Ca2Cinto this intracellular store The ER Ca2Cstore is released on activation of the cell
by stimulants, which results in the production of inositol 1,4,5-trisphosphate (IP3) This sharp increase in cytosolic calcium concentration stimulates the opening of Ca2C channels in the plasma membrane (calcium release activated calcium channels; CRAC channels) allowing Ca2C
to enter the cell down it concentration gradient (calcium release activated calcium current, ICRAC) resulting in a sustained increase in cytosolic Ca2C concentration (Parekh and Penner 1997; Berridge, Bootman, and Lipp 1998; Berridge 2001) A number of the transport proteins involved
in the maintenance of Ca2Chomeostasis are sensitive to oxidative stress For example, the Ca2C -ATPase of the ER contains a cysteine residue that is susceptible to oxidation, as are the IP3receptor calcium channels in the ER
Cytosolic Ca2Ccan be measured using fluorescent dyes such as fura-2 (Grynkiewicz, Poenie, and Tsien 1985) which alter their fluorescent properties on binding to Ca2C Fura-2 has the advantage of being a ratio dye, which permits alterations in background fluorescence, for example due to the introduction of particles, while measuring calcium
Ultrafine carbon black (CB) has been shown to increase the resting cytosolic calcium concen-tration of a human monocytic cell line MonoMac 6 (MM6) (Stone et al 2000) This effect was not
Trang 9observed with the same dose of larger, respirable CB particles or with pathogenic a-quartz (DQ12).
PM10was shown to produce the same type of calcium influx with associated TNFa gene expression (Brown, Donaldson, and Stone 2004b)
Thapsigargin is a useful tool to investigate the potential effects of particles on Ca2Csignaling (Thastrup et al 1990) Thapsigargin works by inhibiting the ER Ca2CATPase, resulting in leak of the ER store contents into the cytosol Treatment with thapsigargin results in a sharp increase in cytosolic Ca2C(comparable to the effect of IP3) followed by a stimulation of the ICRAC Treatment
of a macrophage cell line (MonoMac 6) with ufCB for 30 min induced an increase in the ICRAC
observed on treatment with thapsigargin, through an increased opening of the plasma membrane
Ca2Cchannels (Stone et al 2000) Similar effects were seen in response to PM10—the increase in calcium was involved in TNFa gene expression (Brown, Donaldson, and Stone 2004b)
17.4.6.2 Mitogen-Activated Protein Kinase Cascade
The mitogen-activated protein kinase (MAPK) cascade includes the extra cellular signal-related kinase (ERK1, ERK2) activated in response to growth factors, oxidative stress or phorbol esters via a Ras-dependent mechanism, c-Jun amino terminal kinase/stress activated protein kinase (JNK1, JNK2) activated by TNF-a in a Ras-independent manner and p38 (Seger and Krebs 1995) Activation
of the MAPK cascade involving phosphorylation and dephosphorylation of a number of proteins leads to the transactivation of c-fos and c-jun and a number of interrelated transcription factors (Seger and Krebs 1995) Moreover, in a number of cellular systems, the balance between the activation
of several arms of this pathway appears to govern whether apoptosis or cell proliferation occurs (Xia et al 1995)
Limited studies have been carried out investigating the influence of particulates on the MAPK pathway and these have been reviewed by the authors (Brown et al 2004a; Donaldson et al 2004) One recent study has shown that exposure of normal human bronchial epithelial (NHBE) cells to ultrafine elemental carbon particles induced the phosphorylation and activation of p38 MAPK In addition, inhibition of p38 MAPK activity blocked the interleukin-8 mRNA expression in these cells
17.4.6.3 Transcription Factor Activation
ROS and inflammatory cytokines both cause activation of the transcription factors NF-kB and AP-1 (Meyer, Schreck, and Baeuerle 1993) In addition, ROS have been suggested to act as second messenger molecules within the cell (Sen et al 1997) The transcription factors NF-kB and AP-1 have been shown to be regulated by the intracellular redox status (Piette et al 1997; Ginn-Pease and Whisler 1998) NF-kB is a transcription factor important in the regulation of a number of genes intrinsic to inflammation, proliferation, and lung defenses (Schins and Donaldson 2000) including cytokines, nitric oxide synthase, adhesion molecules, and protooncogenes, such as c-myc The process of NF-kB activation involves the cytoplasmic phosphorylation, ubiquitination and subsequent proteolytic degradation of the IkB inhibitory subunits from kB Release of
NF-kB from INF-kB allows uncovering of the nuclear localization site on the NF-NF-kB subunits so that it can migrate to the nucleus Once in the nucleus, the activated transcription factor complex, which include p65 protein subunits (Donaldson et al 2004), then bind to promoter regions of genes that have consensus NF-kB DNA binding sequences NF-kB has been shown to be activated by
a number of particles (Jimenez et al 2000; Shukla et al 2000; Hubbard et al 2002; McNeilly et al 2005)
AP-1 is a family of accessory transcription factors that interact with other regulatory sequences called TPA-response element (TRE) or AP-1 sites (Donaldson et al 2004) AP-1 transcription factors include homo- (Jun/Jun) and heterodimer (Fos/Jun) complexes encoded by various members of the c-fos and c-jun families of protooncogenes The functional ramifications of c-fos
Trang 10and c-jun transactivation may be cell type specific, but Fos and Jun proteins may regulate the expression of other genes required for the progression through the cell cycle, apoptosis, or cell transformation (Angel and Karin 1991)
A number of different pathogenic particles have been found to activate AP-1 (Timblin, Be´ruBe´, and Mossman 1998; Jimenez et al 2002; Marwick et al 2004; Shukla et al 2004; Brown et al 2004a; McNeilly et al in press)
Activation of transcription factors can be investigated via a number of methods These include the gel mobility shift or retardation assay of transcription factor DNA binding activity, immuno-histochemical analysis of protein localization in cells, gene transactivation assays using reporter gene constructs measuring luciferase activity, and western blotting of protein levels
With growing investigations into the effects of nanoparticles, there is concern that particles might gain access to the blood and thereby exert direct pathogenic effects on the cardiovascular system It
is therefore cogent to discuss testing systems for the effects of particles on the blood
17.4.7.1 Endothelium
The endothelium is an extremely important cell type intimately involved in the regulation of vascular tone, clotting, fibrinolysis, and inflammation, so it is a highly relevant cell to study HUVECs and variants are available that allow the measurement of the effects that particles gaining access to them might have, such as gene expression for clotting factors and inflammatory mediators (Gilmour et al 2005)
17.4.7.2 Platelets
Platelets are a key cell in the initiation of thrombus formation If particles gaining access to the blood could activate platelets, then there would likely be thrombus formation Interactions between platelets and various nanoparticles have been reported (Nemmar et al 2004; Radomski et al 2005) with evidence that some particles can cause platelet aggregation and up-regulation of surface adhesion molecules on the platelets
17.4.7.3 Atherosclerotic Plaques
If particles become blood borne, they are likely to be deposited in the vessel wall in the same way as LDL and by the same forces of turbulence They could then interact directly with the cells in the atherosclerotic lesion There are currently no specific in vitro models to investigate this aspect
Because of interest in the brain translocation of particles, studies of the effects of particles on neurons are warranted These can address the penetration of the blood brain barrier, such as:
1 Evaluation of toxicity leading to increased permeability and opening of tight junctions between endothelial cells If particles in the blood are able to have this effect, they may
be able to leave the blood and traverse the BBB
2 Testing the ability of particles to undergo endocytosis or transcytosis in endothelium of the BBB
3 Assessment of the influence of NP on cell membrane fluidity, which may lead to inhi-bition of the brain efflux system