Long-term toxicity of naturally occurring asbestos in male Fischer 344 rats.

Naturally occurring asbestos (NOA) fibers are found in geologic deposits that may be disturbed by mining, earthworks, or natural processes, resulting in adverse health risks to exposed individuals. The toxicities of Libby amphibole and NOA samples including Sumas Mountain chrysotile (SM), El Dorado tremolite (ED), and Ontario ferroactinolite cleavage fragments (ON) were compared in male Fischer 344 (F344) rats 15 mo after exposure. Rat-respirable fractions of LA and SM displayed greater mean lengths and aspect ratios than ED and ON. After a single intratracheal (IT) instillation (0.5 or 1.5 mg/rat), persistent changes in ventilatory parameters and a significant increase in lung resistance at baseline and after methacholine aerosol dosing were found only in rats exposed to 1.5 mg SM. High-dose ED significantly elevated bronchoalveolar lavage lactate dehydrogenase (LDH) activity and protein levels, while high-dose SM increased γ-glutamyl transferase and LDH activities. A moderate degree of lung interstitial fibrosis after exposure to 1.5 mg SM persisted 15 mo after exposure, unchanged from previous findings at 3 mo. LA induced mild fibrosis, while ED and ON produced minimal and no apparent fibrosis, respectively. Bronchioloalveolar carcinoma was observed 15 mo after exposure to LA or ED. Data demonstrated that SM, given by bolus IT dosing on an equivalent mass basis, induced greater pulmonary function deficits, airway hyperresponsiveness, and interstitial fibrosis than other NOA, although unlike LA and ED, no apparent evidence for carcinogenicity was found. All NOA samples except ON cleavage fragments produced some degree of long-term toxicity.

Comparative long-term toxicity of Libby amphibole and amosite asbestos in rats after single or multiple intratracheal exposures.

In former mine workers of Libby, MT, exposure to amphibole-containing vermiculite was linked to increased rates of asbestosis, lung cancer, and mesothelioma. Although many studies showed adverse effects following exposure to Libby amphibole (LA; a mixture of winchite, richterite, and tremolite), little is known regarding the relative toxicity of LA compared to regulated asbestos, or regarding the risks associated with acute high-dose exposures relative to repeated low-dose exposures. In this study, pulmonary function, inflammation, and pathology were assessed after single or multiple intratracheal (IT) exposures of LA or a well-characterized amosite (AM) control fiber with equivalent fiber characteristics. Male F344 rats were exposed to an equivalent total mass dose (0.15, 0.5, 1.5, or 5 mg/rat) of LA or AM administered either as a single IT instillation, or as multiple IT instillations given every other week over a 13-wk period, and necropsied up to 20 mo after the initial IT. When comparing the two fiber types, in both studies LA resulted in greater acute neutrophilic inflammation and cellular toxicity than equal doses of AM, but long-term histopathological changes were approximately equivalent between fibers, suggesting that LA is at least as toxic as AM. In addition, although no dose-response relationship was discerned, mesothelioma or lung carcinomas were found after exposure to low and high dose levels of LA or AM in both studies. Conversely, when comparing studies, an equal mass dose given over multiple exposures instead of a single bolus resulted in greater chronic pathological changes in lung at lower doses, despite the initially weaker acute inflammatory response. Overall, these results suggest that there is a possibility of greater long-term pathological changes with repeated lower LA dose exposures, which more accurately simulates chronic environmental exposures.

Soy biodiesel emissions have reduced inflammatory effects compared to diesel emissions in healthy and allergic mice.

Toxicity of exhaust from combustion of petroleum diesel (B0), soy-based biodiesel (B100), or a 20% biodiesel/80% petrodiesel mix (B20) was compared in healthy and house dust mite (HDM)-allergic mice. Fuel emissions were diluted to target fine particulate matter (PM(2.5)) concentrations of 50, 150, or 500 µg/m(3). Studies in healthy mice showed greater levels of neutrophils and MIP-2 in bronchoalveolar lavage (BAL) fluid 2 h after a single 4-h exposure to B0 compared with mice exposed to B20 or B100. No consistent differences in BAL cells and biochemistry, or hematological parameters, were observed after 5 d or 4 weeks of exposure to any of the emissions. Air-exposed HDM-allergic mice had significantly increased responsiveness to methacholine aerosol challenge compared with non-allergic mice. Exposure to any of the emissions for 4 weeks did not further increase responsiveness in either non-allergic or HDM-allergic mice, and few parameters of allergic inflammation in BAL fluid were altered. Lung and nasal pathology were not significantly different among B0-, B20-, or B100-exposed groups. In HDM-allergic mice, exposure to B0, but not B20 or B100, significantly increased resting peribronchiolar lymph node cell proliferation and production of T(H)2 cytokines (IL-4, IL-5, and IL-13) and IL-17 in comparison with air-exposed allergic mice. These results suggest that diesel exhaust at a relatively high concentration (500 µg/m(3)) can induce inflammation acutely in healthy mice and exacerbate some components of allergic responses, while comparable concentrations of B20 or B100 soy biodiesel fuels did not elicit responses different from those caused by air exposure alone.

Mice lacking three Loci encoding 14 glutathione transferase genes: a novel tool for assigning function to the GSTP, GSTM, and GSTT families.

Glutathione S-transferases (GSTs) form a superfamily defined by their ability to catalyze the conjugation of glutathione with electrophilic substrates. These enzymes are proposed to play a critical role in protection of cellular components from damage mediated by reactive metabolites. Twenty-two cytosolic GSTs, grouped into seven families, are recognized in mice. This complexity hinders the assignment of function to a subset or family of these genes. We report generation of a mouse line in which the locus encoding three GST gene families is deleted. This includes the four Gstt genes spanning 65 kb on chromosome 10 and the seven Gstm genes found on a 150 kb segment of DNA chromosome 3. In addition, we delete two Gstp genes on chromosome 19 as well as a third related gene located 15 kb telomeric to Gstp1 and Gstp2, which we identify as a potential new member of this gene family. We show that, despite the loss of up to 75% of total GST activity in some tissues from these animals, the mice are healthy and fertile, with normal life expectancy. The normal development and health of these animals make them an appropriate model for defining the role of these families in redox homeostasis and metabolism of drugs and environmental pollutants.

Early and delayed effects of naturally occurring asbestos on serum biomarkers of inflammation and metabolism.

Studies recently showed that intratracheal (IT) instillation of Libby amphibole (LA) increases circulating acute-phase proteins (APP; α-2 macroglobulin, A2M; and α-1 acid glycoprotein, AGP) and inflammatory biomarkers (osteopontin and lipocalin) in rats. In this study, objectives were to (1) compare changes in biomarkers of rats after instillation of different naturally occurring asbestos (NOA) minerals including LA, Sumas Mountain chrysotile (SM), El Dorado Hills tremolite (ED), and Ontario ferroactinolite cleavage fragments (ON), and (2) examine biomarkers after subchronic LA or amosite inhalation exposure. Rat-respirable fractions (aerodynamic diameter approximately 2.5 µm) prepared by water elutriation were delivered via a single IT instillation at doses of 0, 0.5, and 1.5 mg/rat in male F344 rats. Nose-only inhalation exposures were performed at 0, 1, 3.3, and 10 mg/m(3) for LA and at 3.3 mg /m(3) for amosite, 6h/d, 5 d/wk for 13 wk. Inflammation, metabolic syndrome, and cancer biomarkers were analyzed in the serum for up to 18 mo. IT instillation of some asbestos materials significantly increased serum AGP and A2M but to a varying degree (SM = LA > ON = ED). Numerical increases in interleukin (IL)-6 and osteopontin occurred in rats instilled with SM. SM and ED also elevated leptin and insulin at 15 mo, suggesting potential metabolic effects. LA inhalation tended to raise A2M at d 1 but not cytokines. Serum mesothelin appeared to elevate after 18 mo of LA inhalation. These results suggest that the lung injury induced by high levels of asbestos materials may be associated with systemic inflammatory changes and predisposition to insulin resistance.

Allergic inflammation induces a persistent mechanistic switch in thromboxane-mediated airway constriction in the mouse.

Actions of thromboxane (TXA(2)) to alter airway resistance were first identified over 25 years ago. However, the mechanism underlying this physiological response has remained largely undefined. Here we address this question using a novel panel of mice in which expression of the thromboxane receptor (TP) has been genetically manipulated. We show that the response of the airways to TXA(2) is complex: it depends on expression of other G protein-coupled receptors but also on the physiological context of the signal. In the healthy airway, TXA(2)-mediated airway constriction depends on expression of TP receptors by smooth muscle cells. In contrast, in the inflamed lung, the direct actions of TXA(2) on smooth muscle cell TP receptors no longer contribute to bronchoconstriction. Instead, in allergic lung disease, TXA(2)-mediated airway constriction depends on neuronal TP receptors. Furthermore, this mechanistic switch persists long after resolution of pulmonary inflammation. Our findings demonstrate the powerful ability of lung inflammation to modify pathways leading to airway constriction, resulting in persistent changes in mechanisms of airway reactivity to key bronchoconstrictors. Such alterations are likely to shape the pathogenesis of asthmatic lung disease.

Cooperation between mast cells and neurons is essential for antigen-mediated bronchoconstriction.

Mast cells are important sentinels guarding the interface between the environment and the body: a breach in the integrity of this interface can lead to the release of a plethora of mediators that engage the foreign agent, recruit leukocytes, and initiate adaptive physiological changes in the organism. While these capabilities make mast cells critical players in immune defense, it also makes them important contributors to the pathogenesis of diseases such as asthma. Mast cell mediators induce dramatic changes in smooth muscle physiology, and the expression of receptors for these factors by smooth muscle suggests that they act directly to initiate constriction. Contrary to this view, we show herein that mast cell-mediated bronchoconstriction is observed only in animals with intact innervation of the lung and that serotonin release alone is required for this action. While ablation of sensory neurons does not limit bronchoconstriction, constriction after Ag challenge is absent in mice in which the cholinergic pathways are compromised. Linking mast cell function to the cholinergic system likely provides an important means of modulating the function of these resident immune cells to physiology of the lung, but may also provide a safeguard against life-threatening anaphylaxis during mast cell degranulation.

Size Matters: Molecular Weight Specificity of Hyaluronan Effects in Cell Biology.

Hyaluronan signaling properties are unique among other biologically active molecules, that they are apparently not influenced by postsynthetic molecular modification, but by hyaluronan fragment size. This review summarizes the current knowledge about the generation of hyaluronan fragments of different size and size-dependent differences in hyaluronan signaling as well as their downstream biological effects.

Bilateral vagotomy as a tool for determining autonomic involvement in airway responses in mouse models of asthma.

This chapter describes the use of bilateral vagotomy as a tool for determining autonomic regulation of airway responses to the exogenous bronchoconstrictor thromboxane mimetic U46619 in an acute model of asthma in the mouse. Mice receive a sensitization of ovalbumin (OVA) and adjuvant followed by 3 days of OVA aerosol to induce allergic airway disease characterized by bronchoalveolar lavage (BAL) eosinophilia, increased mucus production, and elevated IgE and IL-13. Using a small animal ventilator (Flexi-vent) and the forced oscillatory technique fit to the constant phase model of the lung, a variety of features associated with human asthma can be evaluated in mouse models. For example, this protocol describes the methods to evaluate central and peripheral airway mechanics, airway resistance (R aw) and tissue damping (G), and tissue elastance (H) in response to U46619. The contribution of autonomic nerves in this response is determined by severing both the left and right vagus nerves prior to aerosol challenge.

Sumas Mountain chrysotile induces greater lung fibrosis in Fischer344 rats than Libby amphibole, El Dorado tremolite, and Ontario ferroactinolite.

The physical properties of different types of asbestos may strongly affect health outcomes in exposed individuals. This study was designed to provide understanding of the comparative toxicity of naturally occurring asbestos (NOA) fibers including Libby amphibole (LA), Sumas Mountain chrysotile (SM), El Dorado tremolite (ED), and Ontario ferroactinolite (ON) cleavage fragments. Rat-respirable fractions (PM2.5) were prepared by water elutriation. Surface area was greater for SM (64.1 m²/g) than all other samples (range: 14.1-16.2 m²/g), whereas mean lengths and aspect ratios (ARs) for LA and SM were comparable and greater than ED and ON. Samples were delivered via a single intratracheal (IT) instillation at doses of 0.5 and 1.5mg/rat. One day post-IT instillation, low-dose NOA exposure resulted in a 3- to 4-fold increase in bronchoalveolar lavage fluid (BALF) cellularity compared with dispersion media (DM) controls, whereas high-dose exposure had a more severe effect on lung inflammation which varied by source. Although inducing less neutrophilic inflammation than ON and ED, exposure to either LA or SM resulted in a greater degree of acute lung injury. Three months post-IT instillation, most BALF parameters had returned to control levels, whereas the development of fibrosis persisted and was greatest in SM-exposed rats (SM > LA > ON > ED). These data demonstrate that fiber length and higher AR are directly correlated with the severity of fibrosis and that, in the rat, exposure to SM is more fibrogenic than LA which suggests that there may be cause for concern for people at risk of being exposed to NOA from the Sumas Mountain landslide.