Kent E. Pinkerton

Meeting Report: Hazard Assessment for Nanoparticles—Report from an Interdisciplinary Workshop

In this report we present the findings from a nanotoxicology workshop held 6–7 April 2006 at the Woodrow Wilson International Center for Scholars in Washington, DC. Over 2 days, 26 scientists from government, academia, industry, and nonprofit organizations addressed two specific questions: what information is needed to understand the human health impact of engineered nanoparticles and how is this information best obtained? To assess hazards of nanoparticles in the near-term, most participants noted the need to use existing in vivo toxicologic tests because of their greater familiarity and interpretability. For all types of toxicology tests, the best measures of nanoparticle dose need to be determined. Most participants agreed that a standard set of nanoparticles should be validated by laboratories worldwide and made available for benchmarking tests of other newly created nanoparticles. The group concluded that a battery of tests should be developed to uncover particularly hazardous properties. Given the large number of diverse materials, most participants favored a tiered approach. Over the long term, research aimed at developing a mechanistic understanding of the numerous characteristics that influence nanoparticle toxicity was deemed essential. Predicting the potential toxicity of emerging nanoparticles will require hypothesis-driven research that elucidates how physicochemical parameters influence toxic effects on biological systems. Research needs should be determined in the context of the current availability of testing methods for nanoscale particles. Finally, the group identified general policy and strategic opportunities to accelerate the development and implementation of testing protocols and ensure that the information generated is translated effectively for all stakeholders.

Sidestream Cigarette Smoke Generation and Exposure System for Environmental Tobacco Smoke Studies

AbstractPrevious studies of the effects of environmental tobacco smoke have used artificial systems for generating aged and diluted sidestream cigarette smoke. Generally these systems have been designed for large chambers, which require expensive smoking machines, use large air flows, and are labor-intensive. We present a new method for producing sidestream smoke and describe an exposure system for smaller chambers that collects, ages, and dilutes smoke to simulate environmentally relevant conditions. Furthermore, our system is relatively inexpensive, maintains consistent levels of total suspended particulates (TSP) and carbon mon-oxide that can be set at a variety of concentrations, and significantly reduces the manual component of exposure studies.

Use of Coated Silver Nanoparticles to Understand the Relationship of Particle Dissolution and Bioavailability to Cell and Lung Toxicological Potential

Since more than 30% of consumer products that include engineered nanomaterials contain nano-Ag, the safety of this material is of considerable public concern. In this study, Ag nanoparticles (NPs) are used to demonstrate that 20 nm polyvinylpyrrolidone (PVP or P) and citrate (C)-coated Ag NPs induce more cellular toxicity and oxidative stress than larger (110 nm) particles due to a higher rate of dissolution and Ag bioavailability. Moreover, there is also a higher propensity for citrate 20 nm (C20) nanoparticles to generate acute neutrophilic inflammation in the lung and to produce chemokines compared to C110. P110 has less cytotoxic effects than C110, likely due to the ability of PVP to complex released Ag(+) . In contrast to the more intense acute pulmonary effects of C20, C110 induces mild pulmonary fibrosis at day 21, likely as a result of slow but persistent Ag(+) release leading to a sub-chronic injury response. Interestingly, the released metallic Ag is incorporated into the collagen fibers depositing around airways and the lung interstitium. Taken together, these results demonstrate that size and surface coating affect the cellular toxicity of Ag NPs as well as their acute versus sub-chronic lung injury potential.

Receptor-mediated tobacco toxicity: cooperation of the Ras/Raf-1/MEK1/ERK and JAK-2/STAT-3 pathways downstream of α7 nicotinic receptor in oral keratinocytes

The use of tobacco products is associated with an increased incidence of periodontal disease, poor response to periodontal therapy, and a high risk for developing head and neck cancer. Nicotine and tobacco‐derived nitrosamines have been shown to exhibit their pathobiologic effects due in part to activation of the nicotinic acetylcholine (ACh) receptors (nAChRs), mainly α7 nAChR, expressed by oral keratinocytes (KCs). This study was designed to gain mechanistic insight into α7‐mediated morbidity of tobacco products in the oral cavity. We investigated the signaling pathways downstream of α7 nAChR in monolayers of oral KCs exposed for 24 h to aged and diluted sidestream cigarette smoke (ADSS) or an equivalent concentration of pure nicotine. By both real‐time polymerase chain reaction (PCR) and In‐cell Western, the KCs stimulated with ADSS or nicotine showed multifold increases of STAT‐3. These effects could be completely blocked or significantly P < <0.05) diminished if the cells were pretreated with the α7 antagonist ‐bungarotoxin (BTX) or transfected with anti‐α7 small interfering RNA (siRNA‐α7). The use of pathway inhibitors revealed that signaling through the Ras/Raf‐1/MEK1/ ERK steps mediated 7‐dependent up‐regulation of STAT‐3. Targeted mutation of the α7 gene prevented ERK1/2 activation by nicotine. Using the gel mobility shift assay, we demonstrated that an increased protein binding activity of STAT‐3 caused by ADSS or pure nicotine was mediated by janus‐activated kinase (JAK)‐2. Activation of JAK‐2/STAT‐3 pathway could be prevented by BTX or siRNA‐α7. Thus, nuclear transactivation of STAT‐3 in KCs exposed to tobacco products is mediated via intracellular signaling downstream from α7, which proceeds via two complementary pathways. The Ras/Raf‐1/MEK1/ERK cascade culminates in up‐regulated expression of the gene encoding STAT‐3, whereas recruitment and activation of tyrosine kinase JAK‐2 phosphorylates it. Elucidation of this novel mechanism of nicotine‐dependent nuclear transactivation of STAT‐3 identifies oral α7 nAChR as a promising molecular target to prevent, reverse, or retard tobacco‐related periodontal disease and progression of head and neck cancer by receptor inhibitors.—Arredondo, J., Chernyavsky, A. I., Jolkovsky, D. L., Pinkerton, K. E., Grando, S. A. Receptor‐mediated tobacco toxicity: cooperation of the Ras/Raf‐1/MEK1/ ERK and JAK‐2/STAT‐3 pathways downstream of α7 nicotinic receptor in oral keratinocytes. FASEB J. 20, 2093–2101 (2006)

Health effects of inhaled engineered and incidental nanoparticles

Engineered nanoscale materials provide tremendous promise for technological advancements; however, concerns have been raised about whether research of the possible health risks of these nanomaterials is keeping pace with products going to market. Research on nanomaterials, including carbon nanotubes, semiconductor crystals, and other ultrafine particles (i.e., titanium dioxide, quantum dots, iridium) will be examined to illustrate what is currently known or unknown about how particle characteristics (e.g., size, agglomeration, morphology, solubility, surface chemistry) and exposure/dose metrics (e.g., mass, size, surface area) influence the biological fate and toxicity of inhaled nanosized particles. The fact that nanosized particles (1) have a potentially high efficiency for deposition; (2) target both the upper and lower regions of the respiratory tract; (3) are retained in the lungs for a long period of time, and (4) induce more oxidative stress and cause greater inflammatory effects than their fine-sized equivalents suggest a need to study the impact of these particles on the body. Achieving a better understanding of the dynamics at play between particle physicochemistry, transport patterns, and cellular responses in the lungs and other organs will provide a future basis for establishing predictive measures of toxicity or biocompatibility and a framework for assessing potential human health risks.

Concordance Between In Vitro and In Vivo Dosimetry in the Proinflammatory Effects of Low-Toxicity, Low-Solubility Particles : The Key Role of the Proximal Alveolar Region

We previously demonstrated the importance of the surface area burden as the key dose metric in the elicitation of inflammation in rat lungs by low-solubility, low-toxicity particles (LSLTP). We have now explored the dosimetry of LSLTP in vitro using epithelial cell interleukin (IL)-8 gene expression as a surrogate for potential of particles to cause inflammation. The proximal alveolar region (PAR) of the lung has been identified as a key site for the retention of respirable particles, as it receives high deposition but has slow clearance compared to the larger airways. For these reasons, a few days after exposure to particles the residual dose is concentrated in the PAR region. Re-expressing our rat lung data as particle surface area burden per unit of PAR surface area we obtained a threshold value for onset of inflammation of 1 cm2/cm2. We carried out dose responses in vitro for onset of IL-8 gene expression with the same particles as we had used in vivo. When we expressed the in vitro dose as surface area dose per unit A549cell culture surface area, we obtained a threshold of 1 cm2/cm2. This concordance between proinflammatory effects in vivo (PMN in BAL) and in vitro (epithelial IL-8 gene expression) confirms and supports the utility of the particle surface area metric and the importance of the PAR. These studies also open the way for future in vitro approaches to studying proinflammatory effects of a range of toxic particles based on sound dosimetry that complemenst animal use in particle toxicology.

Interactions between chemical and climate stressors: A role for mechanistic toxicology in assessing climate change risks

Incorporation of global climate change (GCC) effects into assessments of chemical risk and injury requires integrated examinations of chemical and nonchemical stressors. Environmental variables altered by GCC (temperature, precipitation, salinity, pH) can influence the toxicokinetics of chemical absorption, distribution, metabolism, and excretion as well as toxicodynamic interactions between chemicals and target molecules. In addition, GCC challenges processes critical for coping with the external environment (water balance, thermoregulation, nutrition, and the immune, endocrine, and neurological systems), leaving organisms sensitive to even slight perturbations by chemicals when pushed to the limits of their physiological tolerance range. In simplest terms, GCC can make organisms more sensitive to chemical stressors, while alternatively, exposure to chemicals can make organisms more sensitive to GCC stressors. One challenge is to identify potential interactions between nonchemical and chemical stressors affecting key physiological processes in an organism. We employed adverse outcome pathways, constructs depicting linkages between mechanism-based molecular initiating events and impacts on individuals or populations, to assess how chemical- and climate-specific variables interact to lead to adverse outcomes. Case examples are presented for prospective scenarios, hypothesizing potential chemical–GCC interactions, and retrospective scenarios, proposing mechanisms for demonstrated chemical–climate interactions in natural populations. Understanding GCC interactions along adverse outcome pathways facilitates extrapolation between species or other levels of organization, development of hypotheses and focal areas for further research, and improved inputs for risk and resource injury assessments. Environ. Toxicol. Chem. 2013;32:32–48.

California wildfires of 2008: coarse and fine particulate matter toxicity.

Background During the last week of June 2008, central and northern California experienced thousands of forest and brush fires, giving rise to a week of severe fire-related particulate air pollution throughout the region. California experienced PM10–2.5 (particulate matter with mass median aerodynamic diameter > 2.5 μm to < 10 μm; coarse ) and PM2.5 (particulate matter with mass median aerodynamic diameter < 2.5 μm; fine) concentrations greatly in excess of the air quality standards and among the highest values reported at these stations since data have been collected. Objectives These observations prompt a number of questions about the health impact of exposure to elevated levels of PM10–2.5 and PM2.5 and about the specific toxicity of PM arising from wildfires in this region. Methods Toxicity of PM10–2.5 and PM2.5 obtained during the time of peak concentrations of smoke in the air was determined with a mouse bioassay and compared with PM samples collected under normal conditions from the region during the month of June 2007. Results Concentrations of PM were not only higher during the wildfire episodes, but the PM was much more toxic to the lung on an equal weight basis than was PM collected from normal ambient air in the region. Toxicity was manifested as increased neutrophils and protein in lung lavage and by histologic indicators of increased cell influx and edema in the lung. Conclusions We conclude that the wildfire PM contains chemical components toxic to the lung, especially to alveolar macrophages, and they are more toxic to the lung than equal doses of PM collected from ambient air from the same region during a comparable season.

Influence of air pollution on respiratory health during perinatal development

1 The respiratory system is a highly ordered structure composed of over 40 cell types involved in a multitude of functions. Development of the lungs spans from embryogenesis to adult life, passing through several distinct stages of growth. 2 Oxidant gases, airborne particles and environmental tobacco smoke are common air pollutants that could have a significant impact on the lungs during both pre‐ and postnatal periods of life. Although the specific target cells for exposure to these pollutants are not clearly identified, these cells are likely to affect critical signals or mediators expressed during distinct stages of lung development. 3 Neonatal susceptibility to environmental pollutants may be caused by either direct or indirect hits on several cell types to influence cell differentiation, proliferation and/or maturation. Air pollutants may also alter the normal developmental pattern for metabolic, immune and neurological functions that are constantly changing during in utero and postnatal growth. 4 The sensitivity of neonatal cells to environmental insults is likely to be completely different from these same cell types found in the adult. Delivery of an environmental toxicant to the respiratory system is also dramatically different during the fetal compared with the postnatal period. Passage and interaction of environmental factors through other organ systems and the vasculature, as well as maternal influences, must be taken into consideration when evaluating the impact of an environmental toxicant during early life. 5 To understand the heath outcomes of exposure to a variety of environmental factors in the respiratory system of children requires careful consideration that lung development is a multistep process and cannot be based on studies in adults.

Interlaboratory Evaluation of Rodent Pulmonary Responses to Engineered Nanomaterials: The NIEHS Nano GO Consortium

Background: Engineered nanomaterials (ENMs) have potential benefits, but they also present safety concerns for human health. Interlaboratory studies in rodents using standardized protocols are needed to assess ENM toxicity. Methods: Four laboratories evaluated lung responses in C57BL/6 mice to ENMs delivered by oropharyngeal aspiration (OPA), and three labs evaluated Sprague-Dawley (SD) or Fisher 344 (F344) rats following intratracheal instillation (IT). ENMs tested included three forms of titanium dioxide (TiO2) [anatase/rutile spheres (TiO2-P25), anatase spheres (TiO2-A), and anatase nanobelts (TiO2-NBs)] and three forms of multiwalled carbon nanotubes (MWCNTs) [original (O), purified (P), and carboxylic acid “functionalized” (F)]. One day after treatment, bronchoalveolar lavage fluid was collected to determine differential cell counts, lactate dehydrogenase (LDH), and protein. Lungs were fixed for histopathology. Responses were also examined at 7 days (TiO2 forms) and 21 days (MWCNTs) after treatment. Results: TiO2-A, TiO2-P25, and TiO2-NB caused significant neutrophilia in mice at 1 day in three of four labs. TiO2-NB caused neutrophilia in rats at 1 day in two of three labs, and TiO2-P25 and TiO2-A had no significant effect in any of the labs. Inflammation induced by TiO2 in mice and rats resolved by day 7. All MWCNT types caused neutrophilia at 1 day in three of four mouse labs and in all rat labs. Three of four labs observed similar histopathology to O-MWCNTs and TiO2-NBs in mice. Conclusions: ENMs produced similar patterns of neutrophilia and pathology in rats and mice. Although interlaboratory variability was found in the degree of neutrophilia caused by the three types of TiO2 nanoparticles, similar findings of relative potency for the three types of MWCNTs were found across all laboratories, thus providing greater confidence in these interlaboratory comparisons.