Alternate Methods for Assessing Respiratory Toxicity Using Non-animal Methods
Published: December 6, 2007
University of Arizona
Agricultural and Biosystems Engineering
1177 E. 4th Street
Tucson, AZ 85721
Why respiratory toxicity analyses are needed
Numerous epidemiological studies have reported a correlation between exposure to respirable airborne particulate matter (PM) and increased mortality and adverse respiratory health effects, including the development of emphysema, chronic bronchitis, and asthma [Dockery et al., 1992; Pope et al., 1995]. PM is a complex mixture of inorganic and organic compounds whose composition and size depend upon their source. Much of the analyses have focused on coarse particles (with an aerodynamic diameter < 10 µm, also called PM10) and this is the basis of historical U.S. Environmental Protection Agency (EPA) regulations. Fine particles (PM2.5, 0.1 µm – 2.5 µm in aerodynamic diameter) and ultrafine particles (PM0.1, < 100 nm) are considered pathogenic [Oberdörster, 2000] presenting increasing toxicity with decreasing PM aerodynamic diameter. New U.S. EPA regulations address these fine particles, but some have suggested that these new regulations do not go far enough to protect human health [Mitka, 2006]. Only in recent years have the hazards of ultrafines been the subject of much study as they previously had been considered to be of such small mass to be of negligible impact.
It is still not well understood which particles and which chemical components are the primary contributors to the observed health effects. Most likely it is a combination of physicochemical factors including number of particles, particle surface area, surface chemistry (especially the ability to form oxygen radicals), and ability to be internalized by cells of the lung [Donaldson et al., 1998]. Knowledge of the metabolism, disposition, and activation of compounds depositing with or released from PM could allow prediction of the type of cell and tissue damage likely to be caused.
The impact of particles on the lung
On the tissue and cellular level, PM-deposition insults can result in pulmonary inflammation, airway hyperreactivity, epithelial cell damage, and increased epithelial permeability [Fabbri et al., 1984]. The formation of reactive oxygen species and subsequent lipid peroxidation is believed to play a major role in toxicity; however, the rate of formation of reactive oxygen species can depend on synergistic effects between components of PM and on the presence of relatively benign materials. The direct mechanisms by which the wide variety of airborne PM types impact target cells in the respiratory system are diverse, thus severely complicating schemes to monitor the potential impact of the release of such particulates into the atmosphere.
Studies using different in vitro cellular systems have shown varying degrees of proinflammatory and oxidative-stress–related responses after exposure to PM [Brown et al., 2001; Li et al., 2003]. Oxidative stress leading to intracellular damage (often to DNA) and to changes in gene expression and signaling pathways are considered to be the most likely mechanism underlying toxicity of PM. The interpretation of in vitro studies can be difficult as experimenters from differing laboratories utilize their own unique methods for collecting or producing PM, for dosing particles, and for analyzing cellular responses. Standards for testing must be developed.
Epidemiological studies demonstrate a correlation between elevated ambient PM levels and cardiovascular effects. Until recently the causative nature has been unclear. Recent studies have shown that systemic inflammation and increased oxidative stress contribute to the cardiovascular effects [Yang & Ballinger, 2005], but the biological underpinnings of these effects remain unclear.
The types of responses that are observed when toxicity testing procedures are employed will vary based on the type of PM, its concentration, and its mechanism of action. Responses include alterations in cellular metabolic rates, loss of membrane electrical polarity, altered gene expression (leading to secretion of response proteins including cytokines, chemokines, and other inflammatory mediators), changes in cytoarchitecture and adhesion, and initiation of cell death pathways. The metabolism of the epithelial layer lining the lung is often one of the first cellular functions altered by the presence of toxins [Wallaert et al., 2000]. Cell death may proceed through oncotic (gross, uncontrolled death) or apoptotic (programmed cell death) modes. Diesel exhaust particles have been shown to decrease the function of alveolar macrophages [Yin et al., 2007].
Evaluation of PM toxicity is often complicated by synergistic effects or by functional roles for PM components at low concentrations. Metals including Cu, Zn, V, Fe, and Ni are often considered to be amongst the bad actors in PM. However, divalent metals such as Cu and Zn play functional roles in the cell including serving as co-factors in enzymes such as superoxide dismutase [Palmiter & Findley, 1995]. Zn impacts cellular differentiation, apoptosis, and cellular proliferation and so is normally present at intracellular concentrations as high as 0.2 mM [Palmiter & Findley, 1995]. In some cells, the uptake of Cu and Zn appears to occur through passive mechanisms, not involving specific transporters [Ferruzza et al., 2000], which is followed by sequestration of the metals in intracellular vesicles. These metals may be exported from the cell by plasma membrane transporters [Haase & Beyersmann, 1999]. Alternatively, metals may bind to cysteine or histidine residues of a number of storage proteins including metallothionien or metal-binding chaperones [Haase & Beyersmann, 1999]. Sequestration of metals by such storage mechanisms provides some degree of protection to the cell from negative effects caused by the ability of metals to promote formation of highly reactive oxygen species or to displace other metal cofactors from their natural ligands.
The impact of nano-scale materials on the respiratory system is becoming an increasingly important topic due to the increased use of such materials in commercial products. Although the background mass of ambient air nanoparticles is very low (0.5–2 µg/m3), it can increase several-fold during high pollution episodes or in close proximity to highways [Oberdörster et al., 2005]. It is not known what effects these compounds will have; however, their small size (et al., 2005]. Often, manufactured nanoparticles are produced with a surface coating designed to increase their reactivity or to improve the ability for the particles to be suspended in water. These coatings are likely to increase the inherent toxicity.
Challenges and limitation of animal studies
Most importantly for toxicity analyses, a balance must be struck between assessing particle exposure loads with analyses of particle composition and biological activity. Measurement of particle mass and diameter (as currently performed as per US EPA regulations) misses the important aspects of composition and biological activity. A “canary in a coal mine” is needed in which the canary is replaced by some biological entity that can provide a reproducible and consistent response to potential hazards. The issues of particle mass and diameter on deposition can be addressed mechanically.
In vivo studies are considered the most useful toxicological analyses of inhalation hazards since they provide a physiologically relevant response that can be used for predicting the impact of PM on human health. The primary advantage of in vitro studies is that unintended consequences can be assessed. For example, the impact of particulates on non-respiratory tissue can be assessed. The current differences between in vivo and in vitro testing of respiratory hazards can be (grossly) simplified as differences in the delivery of particles and in the biological responses.
The scientific reasons for moving away from animal studies include their extreme cost and time, which lead to a very low sample throughput, and the difficulties in extrapolating across species. There are significant problems in extrapolating high-dose animal studies to low dose chronic exposures since the capacity of the airway epithelium to deactivate compounds can be saturated at the high concentrations and short times often tested on laboratory animals [Gerde, 2005].
Delivery of particulates can be represented and controlled to mimic the human in vivo situation using mechanical systems such as virtual impactors, which permit collection and delivery of a precise mass and particle size. One of the primary limitations in assessing particle loads lies in matching the type and amount of respirable particulate exposure encountered by a person at home, in a work environment, or outside. This can be assessed using small, non-intrusive wearable particle collectors and monitors.
Correlations have been established relating PM toxicity with the concentration of certain metals [Adamson et al., 2000]; however, the analysis cannot be performed using a simple additive approach. Whereas Dye and coworkers  demonstrated that epithelial cell response to residual oil fly ash correlated strongly with the ash content of V, but not with Fe or Ni. A strong correlation has been found between V and Fe levels and cellular toxicity, whereas correlation between Zn content and toxicity can be poor [Okeson et al., 2003]. Part of this difficulty in developing correlations is likely due to synergistic effects between metals. For example, Zn, in moderate concentrations, can mitigate the effects of V and Cu, but not Ni or Fe [Riley et al., 2003]. Such difficulties in developing clear correlations between PM composition and ultimately with toxicity in humans motivates the development of biological activity based monitoring schemes.
The capability of permitting development of synergist effects can be mimicked through more complex design of cell culture, in vitro studies. The major challenge lies in assessing the full array of information provided by the exposed (respiratory) tissue. These include release of signaling compounds (cytokines, reactive oxygen species, etc.), permissive transport of the particles (crossing a cell barrier), and physical changes (loss of lung compliance, loss of tight cell-cell connections, and inactivation or death). These attributes can be quantified in cell culture methods with the proper experimental design. The challenge is that researchers have not been asking such holistic questions in a manner that readily leads to well engineered experimental systems.
A significant challenge that has not yet been fully met by in vitro systems is the translocation of respired particles to other tissues. For example, Elder and coworkers have shown that ultrafine particles comprised of manganese oxide inhaled nasally by monkeys do not cause lung inflammation but accumulated in the olfactory bulb [Elder et al., 2006]. They concluded that the olfactory neuronal pathway was efficient at translocating ultrafine particles to the central nervous system. Such unintended consequences can be assessed by analysis of particle transport across cellular monolayers which can be performed with a higher degree of experimental control in vitro than with in vivo tests.
A critical need for testing of PM (and especially of nanoparticles) needs to address the presence of co-depositing materials. Exposures to PM do not occur in isolation, but arise in the presence of multiple types (size and composition) of PM along with volatile and low-volatile toxicants and biogenic compounds. The interactive effects are infrequently assessed but are likely to play a role in the observable effects. Utilizing cell cultures and analogues allows testing of the enormous number of combinations of experimental conditions required to fully understand these effects.
There are significant differences that arise between in vivo and in vitro systems responding to xenobiotic compounds including benzo(a)pyrene and paraquat. In some cases, cell culture systems cannot provide the necessary response mechanisms due to low or nonexistent expression of transformative enzymes. For example, A549 cells (derived from a human lung adnocarcinoma) are appropriate to investigate the mechanism of active toxins, but compounds that have to be biotransformed by P450 enzymes prior to eliciting toxicity cannot be properly assessed since A549 cells produce lower levels of xenobiotic metabolizing enzymes [Castell et al., 2005].
Approaches for cell culture testing
A highly innovative approach that utilizes cell cultures to circumvent the limitation of a single cell type includes cell analogues of physiologically based pharmacokinetic (PBPK) models. A PBPK typically is a computer model that mimics interactions of a potential toxin with multiple tissue types and accounts for the dynamics of tissue residence and biotransformation. Such models can be used in conjunction with and to design more robust cell culture methods.
Cell culture analogues have been constructed as experimental systems in which co-cultures of organ constituents (liver, kidney, muscle) are fluidically connected [Sweeney et al., 1995]. Tissues will potentially experience the same dynamic exposure that would occur within whole animals thus permitting evaluation of unsuspected interaction. Viravaidya and Shuler  have developed a microscale cell culture analog device for testing of toxicity and bioaccumulation. This consists of a four-chambers representing lung, liver, fat, and a more general tissue. Such methods require refinement before they are able to provide an appropriate test bed for analysis of inhalation toxicity hazards and all of the responses that they may generate.
The recognition obtained from in vitro studies of the potential mechanisms of damage caused by PM can be utilized to develop cell culture analogs to assess each mechanism in a more thorough manner. For example, the translocation of ultrafine particles [Elder et al., 2005] can be evaluated using transport studies of movement across or accumulation in cell monolayers. Similar designs can be developed to assess cellular function or other complex responses.
In summary, toxicity screening was once performed exclusively with whole animal studies. In recent years, cell cultures have gained acceptance as a first step in this screening process. In the short term, in vitro cell cultures will likely not replace the need for animal toxicity studies entirely due to the whole animal response. However, developments in cell culture analogues and in selecting appropriate metrics of toxicity can significantly reduce the number of in vivo studies required.
©2007 Mark Riley
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