Jack R. Harkema

Increased airway epithelial Na + absorption produces cystic fibrosis-like lung disease in mice

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene result in defective epithelial cAMP-dependent Cl− secretion and increased airway Na+ absorption. The mechanistic links between these altered ion transport processes and the pathogenesis of cystic fibrosis lung disease, however, are unclear. To test the hypothesis that accelerated Na+ transport alone can produce cystic fibrosis-like lung disease, we generated mice with airway-specific overexpression of epithelial Na+ channels (ENaC). Here we show that increased airway Na+ absorption in vivo caused airway surface liquid (ASL) volume depletion, increased mucus concentration, delayed mucus transport and mucus adhesion to airway surfaces. Defective mucus transport caused a severe spontaneous lung disease sharing features with cystic fibrosis, including mucus obstruction, goblet cell metaplasia, neutrophilic inflammation and poor bacterial clearance. We conclude that increasing airway Na+ absorption initiates cystic fibrosis-like lung disease and produces a model for the study of the pathogenesis and therapy of this disease.

Ambient Particulate Pollutants in the Ultrafine Range Promote Early Atherosclerosis and Systemic Oxidative Stress

Air pollution is associated with significant adverse health effects, including increased cardiovascular morbidity and mortality. Exposure to particulate matter with an aerodynamic diameter of <2.5 &mgr;m (PM2.5) increases ischemic cardiovascular events and promotes atherosclerosis. Moreover, there is increasing evidence that the smallest pollutant particles pose the greatest danger because of their high content of organic chemicals and prooxidative potential. To test this hypothesis, we compared the proatherogenic effects of ambient particles of <0.18 &mgr;m (ultrafine particles) with particles of <2.5 &mgr;m in genetically susceptible (apolipoprotein E–deficient) mice. These animals were exposed to concentrated ultrafine particles, concentrated particles of <2.5 &mgr;m, or filtered air in a mobile animal facility close to a Los Angeles freeway. Ultrafine particle–exposed mice exhibited significantly larger early atherosclerotic lesions than mice exposed to PM2.5 or filtered air. Exposure to ultrafine particles also resulted in an inhibition of the antiinflammatory capacity of plasma high-density lipoprotein and greater systemic oxidative stress as evidenced by a significant increase in hepatic malondialdehyde levels and upregulation of Nrf2-regulated antioxidant genes. We conclude that ultrafine particles concentrate the proatherogenic effects of ambient PM and may constitute a significant cardiovascular risk factor.

Insights Into the Mechanisms and Mediators of the Effects of Air Pollution Exposure on Blood Pressure and Vascular Function in Healthy Humans

Fine particulate matter air pollution plus ozone impairs vascular function and raises diastolic blood pressure. We aimed to determine the mechanism and air pollutant responsible. The effects of pollution on heart rate variability, blood pressure, biomarkers, and brachial flow-mediated dilatation were determined in 2 randomized, double-blind, crossover studies. In Ann Arbor, 50 subjects were exposed to fine particles (150 &mgr;g/m3) plus ozone (120 parts per billion) for 2 hours on 3 occasions with pretreatments of an endothelin antagonist (Bosentan, 250 mg), antioxidant (Vitamin C, 2 g), or placebo. In Toronto, 31 subjects were exposed to 4 different conditions (particles plus ozone, particles, ozone, and filtered air). In Toronto, diastolic blood pressure significantly increased (2.9 and 3.6 mm Hg) only during particle-containing exposures in association with particulate matter concentration and reductions in heart rate variability. Flow-mediated dilatation significantly decreased (2.0% and 2.9%) only 24 hours after particle-containing exposures in association with particulate matter concentration and increases in blood tumor necrosis factor &agr;. In Ann Arbor, diastolic blood pressure significantly similarly increased during all of the exposures (2.5 to 4.0 mm Hg), a response not mitigated by pretreatments. Flow-mediated dilatation remained unaltered. Particulate matter, not ozone, was responsible for increasing diastolic blood pressure during air pollution inhalation, most plausibly by instigating acute autonomic imbalance. Only particles from urban Toronto additionally impaired endothelial function, likely via slower proinflammatory pathways. Our findings demonstrate credible mechanisms whereby fine particulate matter could trigger acute cardiovascular events and that aspects of exposure location may be an important determinant of the health consequences.

The Nose Revisited: A Brief Review of the Comparative Structure, Function, and Toxicologic Pathology of the Nasal Epithelium

The nose is a very complex organ with multiple functions that include not only olfaction, but also the conditioning (e.g., humidifying, warming, and filtering) of inhaled air. The nose is also a “scrubbing tower” that removes inhaled chemicals that may be harmful to the more sensitive tissues in the lower tracheobronchial airways and pulmonary parenchyma. Because the nasal airway may also be a prime target for many inhaled toxicants, it is important to understand the comparative aspects of nasal structure and function among laboratory animals commonly used in inhalation toxicology studies, and how nasal tissues and cells in these mammalian species may respond to inhaled toxicants. The surface epithelium lining the nasal passages is often the first tissue in the nose to be directly injured by inhaled toxicants. Five morphologically and functionally distinct epithelia line the mammalian nasal passages—olfactory, respiratory, squamous, transitional, and lymphoepithelial—and each nasal epithelium may be injured by an inhaled toxicant. Toxicant-induced epithelial lesions in the nasal passages of laboratory animals (and humans) are often site-specific and dependent on the intranasal regional dose of the inhaled chemical and the sensitivity of the nasal epithelial tissue to the specific chemical. In this brief review, we present examples of nonneoplastic epithelial lesions (e.g., cell death, hyperplasia, metaplasia) caused by single or repeated exposure to various inhaled chemical toxicants. In addition, we provide examples of how nasal maps may be used to record the character, magnitude and distribution of toxicant-induced epithelial injury in the nasal airways of laboratory animals. Intranasal mapping of nasal histopathology (or molecular and biochemical alterations to the nasal mucosa) may be used along with innovative dosimetric models to determine dose/response relationships and to understand if site-specific lesions are driven primarily by airflow, by tissue sensitivity, or by another mechanism of toxicity. The present review provides a brief overview of comparative nasal structure, function and toxicologic pathology of the mammalian nasal epithelium and a brief discussion on how data from animal toxicology studies have been used to estimate the risk of inhaled chemicals to human health.

The adjuvant effect of ambient particulate matter is closely reflected by the particulate oxidant potential

Background It has been demonstrated that ambient particulate matter (PM) can act as an adjuvant for allergic sensitization. Redox-active organic chemicals on the particle surface play an important role in PM adverse health effects and may determine the adjuvant effect of different particle types according to their potential to perturb redox equilibrium in the immune system. Objectives We determined whether the adjuvant effect of ambient fine particles versus ultrafine particles (UFPs) is correlated to their prooxidant potential. Methods We have established an intranasal sensitization model that uses ambient PM as a potential adjuvant for sensitization to ovalbumin (OVA), which enhances the capacity for secondary OVA challenge to induce allergic airway inflammation. Results UFPs with a greater polycyclic aromatic hydrocarbon (PAH) content and higher oxidant potential enhanced OVA sensitization more readily than did fine particles. This manifests as enhanced allergic inflammation upon secondary OVA challenge, leading to eosinophilic inflammation and mucoid hyperplasia starting at the nasal turbinates all the way down to the small pulmonary airways. The thiol antioxidant N-acetyl cysteine was able to suppress some of these sensitization events. Conclusions The adjuvant effects of ambient UFP is determined by their oxidant potential, which likely plays a role in changing the redox equilibrium in the mucosal immune system.

Proliferative and Nonproliferative Lesions of the Rat and Mouse Respiratory Tract

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally-accepted nomenclature for proliferative and non-proliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the respiratory tract of laboratory rats and mice, with color photomicrographs illustrating examples of some lesions. The standardized nomenclature presented in this document is also available electronically on the inter-net (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous developmental and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for respiratory tract lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Production of Interferon-γ by Influenza Hemagglutinin-Specific CD8 Effector T Cells Influences the Development of Pulmonary Immunopathology

This study examined the inflammation, lung function impairment, and immune protection associated with either wild-type or interferon (IFN)-gamma-deficient Tc1- or Tc2-CD8 effector cells responding to influenza pneumonia. The adoptive transfer of influenza hemagglutinin-specific Tc1 effectors afforded protection and elicited only minimal impairment of lung function. IFN-gamma-deficient Tc1 effector cells were equally protective, but were associated with an eosinophil influx and slightly more lung function impairment early in the response. Relative to Tc1, Tc2 effector cells were less protective, elicited an eosinophil influx and a greater impairment of lung functions. IFN-gamma-deficient Tc2 effector cells were not protective and were associated with the severest impairment of lung function throughout the response, an accumulation of neutrophils, and extensive pulmonary vasculitis and alveolar hemorrhaging. Deletion of IFN-gamma was associated with a delay in effector cell recruitment and the elicitation of a more intense inflammatory response that resulted in more severe lung function impairment in the recipients of either Tc1 or Tc2 IFN-gamma-deficient effector cells. Thus, during influenza infections, IFN-gamma production by the responding CD8 T cells is associated with effector cell recruitment and mitigation of the associated inflammation and of the resulting impairment in lung functions but is not necessary for optimal protection.

Nanotoxicology—A Pathologist’s Perspective

Advances in chemistry and engineering have created a new technology, nanotechnology, involving the tiniest known manufactured products. These products have a rapidly increasing market share and appear poised to revolutionize engineering, cosmetics, and medicine. Unfortunately, nanotoxicology, the study of nanoparticulate health effects, lags behind advances in nanotechnology. Over the past decade, existing literature on ultrafine particles and respirable durable fibers has been supplemented by studies of first-generation nanotechnology products. These studies suggest that nanosizing increases the toxicity of many particulates. First, as size decreases, surface area increases, thereby speeding up dissolution of soluble particulates and exposing more of the reactive surface of durable but reactive particulates. Second, nanosizing facilitates movement of particulates across cellular and intracellular barriers. Third, nanosizing allows particulates to interact with, and sometimes even hybridize with, subcellular structures, including in some cases microtubules and DNA. Finally, nanosizing of some particulates, increases pathologic and physiologic responses, including inflammation, fibrosis, allergic responses, genotoxicity, and carcinogenicity, and may alter cardiovascular and lymphatic function. Knowing how the size and physiochemical properties of nanoparticulates affect bioactivity is important in assuring that the exciting new products of nanotechnology are used safely. This review provides an introduction to the pathology and toxicology of nanoparticulates.

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.

AMPLIFIED PROINFLAMMATORY CYTOKINE EXPRESSION AND TOXICITY IN MICE COEXPOSED TO LIPOPOLYSACCHARIDE AND THE TRICHOTHECENE VOMITOXIN (DEOXYNIVALENOL)

A single oral exposure to the trichothecene vomitoxin (VT) has been previously shown in the mouse to increase splenic mRNA levels for several cytokines in as little as 2 h. Since one underlying mechanism for these effects likely involves superinduction of transiently expressed cytokine genes, VT may also potentially amplify cytokine responses to inflammatory stimuli. To test this possibility, the effects of oral VT exposure on tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-1beta expression were measured in mice that were intraperitoneally injected with lipopolysaccharide (LPS), a prototypic inflammatory agent. As anticipated, VT alone at 1, 5, and 25 mg/kg body weight increased splenic mRNA expression of all three cytokines after 3 h in a dose-response fashion. LPS injection at 1 and 5 mg/kg body weight also induced proinflammatory cytokine mRNA expression. There was a synergistic increase in TNF-alpha splenic mRNA levels in mice treated with both VT and LPS as compared to mice treated with either toxin alone, whereas the effects were additive for IL-6 and IL-1beta mRNA expression. When relative mRNA levels were examined over a 12-h period in mice given LPS (1 mg/kg) and/or VT (5 mg/kg), significant enhancement was observed up to 6, 12, and 3 h for TNF-alpha, IL-6, and IL-1beta, respectively. When plasma cytokine concentrations were measured, TNF-alpha was found to peak at 1 h and was significantly increased at 1, 3, and 6 h if mice were given LPS and VT, whereas LPS or VT alone caused much smaller increases in plasma TNF-alpha Plasma IL-6 peaked at 3 h in LPS, VT, and LPS/VT groups, with the combined toxin group exhibiting additive effects. Plasma IL-1beta was not detectable. The potential for VT and LPS to enhance toxicity was examined in a subsequent study. Mortality was not observed up to 72 h in mice exposed to a single oral dose of VT at 25 mg/kg body weight or to an intraperitoneal dose of LPS at 1 or 5 mg/kg body weight; however, all mice receiving VT and either LPS dose became moribund in less than 40 h. The principal histologic lesions in the moribund mice treated with VT and LPS were marked cell death and loss in thymus, Peyers patches, spleen, and bone marrow. In all of these lymphoid tissues, treatment-induced cell death had characteristic histologic features of apoptosis causing lymphoid atrophy. These results suggest that LPS exposure may markedly increase the toxicity of trichothecenes and that the immune system was a primary target of these interactive effects.