R. Clark Lantz

Adverse respiratory effects following overhaul in firefighters.

Overhaul is the stage in which firefighters search for and extinguish possible sources of reignition. It is common practice not to wear respiratory protection during overhaul. Fifty-one firefighters in two groups, 25 without respiratory protection and 26 wearing cartridge respirators, were monitored for exposure to products of combustion and changes in spirometric measurements and lung permeability following overhaul of a structural fire. Testing at baseline and 1 hour after overhaul included forced vital capacity (FVC), forced expiratory volume in one second (FEV1), serum Clara cell protein (CC16), and serum surfactant-associated protein A (SP-A). Overhaul increased CC16 in both groups, indicating increased alveolar-capillary membrane permeability. Contrary to expectations, SP-A increased and FVC and FEV1 decreased in the firefighters wearing cartridge respirators. Changes in FEV1, CC16, and SP-A were associated with concentrations of specific products of combustion or carboxyhemoglobin levels. Firefighter exposures during overhaul have the potential to cause changes in spirometric measurements and lung permeability, and self-contained breathing apparatus should be worn during overhaul to prevent lung injury.

Role of Oxidative Stress in Arsenic-Induced Toxicity

Arsenic is recognized as a carcinogen for human skin, bladder, and lung, following either ingestion or inhalation; however the exact mode of action of environmentally relevant exposure has not been determined. Because arsenic in the environment exists in several oxidative states and can interact with thiols, it is thought that arsenic toxicity is mediated through oxidative stress. Production of oxygen radicals following acute in vitro exposures has been demonstrated. However, our research has chosen to focus on the role of oxidative stress following whole animal exposure to environmentally relevant doses of arsenic. Following a 28-d inhalation of arsenic or cigarette smoke or both, there was a significant decrease in both the reduced and total glutathione levels in the combined arsenic and smoke group compared to groups exposed to arsenic or smoke alone. This correlated with a 5-fold increase in DNA oxidation. Lungs processed for immunohistochemistry localization of 8-oxo-dG showed increased staining in nuclei of airway epithelium and subadjacent interstitial cells. Increases in DNA oxidation were not due to increased inflammation. Although inhalation of arsenic is an important occupational exposure, the majority of human exposures occurs through ingestion of arsenic. Our recent work has been devoted to the identification of altered pulmonary gene and protein expression following ingestion of environmentally relevant levels of arsenic in drinking water. We have found that, following chronic exposure, arsenic leads to misregulation of a number of genes and proteins in the lung. A large percentage of the altered genes and proteins are known to be regulated by redox-sensitive transcription factors, (SP1, NF κB, AP-1), suggesting that, at environmentally relevant levels of chronic exposure, arsenic may be acting through alteration of cellular redox status. Validation of the alterations seen in animal models of exposure is being carried out in humans.

Arsenic toxicology: translating between experimental models and human pathology.

Background: Chronic arsenic exposure is a worldwide health problem. How arsenic exposure promotes a variety of diseases is poorly understood, and specific relationships between experimental and human exposures are not established. We propose phenotypic anchoring as a means to unify experimental observations and disease outcomes. Objectives: We examined the use of phenotypic anchors to translate experimental data to human pathology and investigated research needs for which phenotypic anchors need to be developed. Methods: During a workshop, we discussed experimental systems investigating arsenic dose/exposure and phenotypic expression relationships and human disease responses to chronic arsenic exposure and identified knowledge gaps. In a literature review, we identified areas where data exist to support phenotypic anchoring of experimental results to pathologies from specific human exposures. Discussion: Disease outcome is likely dependent on cell-type–specific responses and interaction with individual genetics, other toxicants, and infectious agents. Potential phenotypic anchors include target tissue dosimetry, gene expression and epigenetic profiles, and tissue biomarkers. Conclusions: Translation to human populations requires more extensive profiling of human samples along with high-quality dosimetry. Anchoring results by gene expression and epigenetic profiling has great promise for data unification. Genetic predisposition of individuals affects disease outcome. Interactions with infectious agents, particularly viruses, may explain some species-specific differences between human pathologies and experimental animal pathologies. Invertebrate systems amenable to genetic manipulation offer potential for elaborating impacts of specific biochemical pathways. Anchoring experimental results to specific human exposures will accelerate understanding 
of mechanisms of arsenic-induced human disease.

Tanshinone I Activates the Nrf2-Dependent Antioxidant Response and Protects Against As(III)-Induced Lung Inflammation In Vitro and In Vivo

AIMS The NF-E2 p45-related factor 2 (Nrf2) signaling pathway regulates the cellular antioxidant response and activation of Nrf2 has recently been shown to limit tissue damage from exposure to environmental toxicants, including As(III). In an attempt to identify improved molecular agents for systemic protection against environmental insults, we have focused on the identification of novel medicinal plant-derived Nrf2 activators. RESULTS Tanshinones [tanshinone I (T-I), tanshinone IIA, dihydrotanshinone, cryptotanshinone], phenanthrenequinone-based redox therapeutics derived from the medicinal herb Salvia miltiorrhiza, have been tested as experimental therapeutics for Nrf2-dependent cytoprotection. Using a dual luciferase reporter assay overexpressing wild-type or mutant Kelch-like ECH-associated protein-1 (Keap1), we demonstrate that T-I is a potent Keap1-C151-dependent Nrf2 activator that stabilizes Nrf2 by hindering its ubiquitination. In human bronchial epithelial cells exposed to As(III), T-I displays pronounced cytoprotective activity with upregulation of Nrf2-orchestrated gene expression. In Nrf2 wild-type mice, systemic administration of T-I attenuates As(III) induced inflammatory lung damage, a protective effect not observed in Nrf2 knockout mice. INNOVATION Tanshinones have been identified as a novel class of Nrf2-inducers for antioxidant tissue protection in an in vivo As(III) inhalation model, that is relevant to low doses of environmental exposure. CONCLUSION T-I represents a prototype Nrf2-activator that displays cytoprotective activity upon systemic administration targeting lung damage originating from environmental insults. T-I based Nrf2-directed systemic intervention may provide therapeutic benefit in protecting other organs against environmental insults.

Pulmonary Biomarkers Based on Alterations in Protein Expression after Exposure to Arsenic

Objective Environmental exposure to arsenic results in multiple adverse effects in the lung. Our objective was to identify potential pulmonary protein biomarkers in the lung-lining fluid of mice chronically exposed to low-dose As and to validate these protein changes in human populations exposed to As. Methods Mice were administered 10 or 50 ppb As (sodium arsenite) in their drinking water for 4 weeks. Proteins in the lung-lining fluid were identified using two-dimensional gel electrophoresis (n = 3) or multidimensional protein identification technology (MUDPIT) (n = 2) coupled with mass spectrometry. Lung-induced sputum samples were collected from 57 individuals (tap water As ranged from ~ 5 to 20 ppb). Protein levels in sputum were determined by ELISA, and As species were analyzed in first morning void urine. Results Proteins in mouse lung-lining fluid whose expression was consistently altered by As included glutathione-S-transferase (GST)-omega-1, contraspin, apolipoprotein A-I and A-IV, enolase-1, peroxiredoxin-6, and receptor for advanced glycation end products (RAGE). Validation of the putative biomarkers was carried out by evaluating As-induced alterations in RAGE in humans. Regression analysis demonstrated a significant negative correlation (p = 0.016) between sputum levels of RAGE and total urinary inorganic As, similar to results seen in our animal model. Conclusion Combinations of proteomic analyses of animal models followed by specific analysis of human samples provide an unbiased determination of important, previously unidentified putative biomarkers that may be related to human disease.

Short-Term Pulmonary Response to Inhaled JP-8 Jet Fuel Aerosol in Mice

B6.A.D. (Ahrd/Nat s ) mice were utilized to investigate the short-term pulmonary response to JP-8 jet fuel (JP-8) aerosol inhalation. Mice were nose-only exposed to atmospheres of 0 to 118 mg/m3 for 1 h/d over a period of 7 days to further test the hypothesis that JP-8 concentrations below the permissible exposure level (PEL) of 350 mg/m3 will induce lung injury. At 24 to 30 hours after the final exposure, pulmonary function and respiratory permeability were measured on anesthetized mice and then randomly assigned for bronchoalveolar lavage or histopathology. Bronchoalveolar lavage fluid (BALF) was analyzed for total protein, lactic dehydrogenase (LDH), N-acetyl-β-D-glucosaminidase (NAG), and cytology. Respiratory permeability increases were observed following doses of 48 and 118 mg/m3 and were supported by concomitant BALF increases in total protein and LDH. Conversely, NAG and alveolar macrophage levels decreased following the same exposure concentrations. Morphological lung injury was characterized by the targeting of bronchiolar epithelium and consisted of perivascular edema, Clara cell vacuolization, and necrosis. Alveolar injury included sporadic pulmonary edema, intra-alveolar hemorrhage, and alterations in type II epithelial cells. These results indicate that repeated inhalation of aerosolized JP-8 induces physiological, biochemical, cellular, and morphological lung injury. This study also provides evidence for the reevaluation of the 350 mg/m3 PEL for more volatile petroleum distillates with regard to respirable aerosols.

In utero and postnatal exposure to arsenic alters pulmonary structure and function.

In addition to cancer endpoints, arsenic exposures can also lead to non-cancerous chronic lung disease. Exposures during sensitive developmental time points can contribute to the adult disease. Using a mouse model, in utero and early postnatal exposures to arsenic (100 ppb or less in drinking water) were found to alter airway reactivity to methacholine challenge in 28 day old pups. Removal of mice from arsenic exposure 28 days after birth did not reverse the alterations in sensitivity to methacholine. In addition, adult mice exposed to similar levels of arsenic in drinking water did not show alterations. Therefore, alterations in airway reactivity were irreversible and specific to exposures during lung development. These functional changes correlated with protein and gene expression changes as well as morphological structural changes around the airways. Arsenic increased the whole lung levels of smooth muscle actin in a dose dependent manner. The level of smooth muscle mass around airways was increased with arsenic exposure, especially around airways smaller than 100 microm in diameter. This increase in smooth muscle was associated with alterations in extracellular matrix (collagen, elastin) expression. This model system demonstrates that in utero and postnatal exposure to environmentally relevant levels of arsenic can irreversibly alter pulmonary structure and function in the adults.

Arsenic and Cigarette Smoke Synergistically Increase DNA Oxidation in the Lung

Epidemiological evidence has indicated that arsenic and cigarette smoking exposure act synergistically to increase the incidence of lung cancer. Since oxidative damage of DNA has been linked to cancer, our hypothesis is that aerosolized arsenic and cigarette smoke work synergistically to increase oxidative stress and increase DNA oxidation in the lung. To test this hypothesis male Syrian golden hamsters were exposed to room air (control), aerosolized arsenic compounds (3.2 mg/m3 for 30 minutes), cigarette smoke (5 mg/m3 for 30 minutes), or both smoke and arsenic. Exposures were for 5 days/week for 5 or 28-days. Animals were sacrificed one day after the last exposure. In the 28-day group, glutathione levels and DNA oxidation (8-oxo-2′-deoxyguanosine (8-oxo-dG)) were determined. Our results show that in the 28-day arsenic/smoke group there was a significant decrease in both the reduced and total glutathione levels compared with arsenic or smoke alone. This correlated with a 5-fold increase in DNA oxidation as shown by HPLC. Immunohistochemical localization of 8-oxo-dG showed increase staining in nuclei of airway epithelium and subadjacent interstitial cells. These results show that dual exposure of arsenic and cigarette smoke at environmentally relevant levels can act synergistically to cause DNA damage.

Arsenic upregulates MMP-9 and inhibits wound repair in human airway epithelial cells

As part of the innate immune defense, the polarized conducting lung epithelium acts as a barrier to keep particulates carried in respiration from underlying tissue. Arsenic is a metalloid toxicant that can affect the lung via inhalation or ingestion. We have recently shown that chronic exposure of mice or humans to arsenic (10-50 ppb) in drinking water alters bronchiolar lavage or sputum proteins consistent with reduced epithelial cell migration and wound repair in the airway. In this report, we used an in vitro model to examine effects of acute exposure of arsenic (15-290 ppb) on conducting airway lung epithelium. We found that arsenic at concentrations as low as 30 ppb inhibits reformation of the epithelial monolayer following scrape wounds of monolayer cultures. In an effort to understand functional contributions to epithelial wound repair altered by arsenic, we showed that acute arsenic exposure increases activity and expression of matrix metalloproteinase (MMP)-9, an important protease in lung function. Furthermore, inhibition of MMP-9 in arsenic-treated cells improved wound repair. We propose that arsenic in the airway can alter the airway epithelial barrier by restricting proper wound repair in part through the upregulation of MMP-9 by lung epithelial cells.

Neutral Endopeptidase (NEP) and Its Role in Pathological Pulmonary Change With Inhalation Exposure To JP-8 Jet Fuel

Through a simulated flightline exposure protocol, Fischer 344 rats (F344) were subjected to an aerosol/vapor mix of the military jet fuel, JP-8. Previous studies with this model of lung injury have revealed significant increases in pulmonary resistance, increased alveolar clearance of 99mTcDTPA, and a decrease in bronchoalveolar lavage fluid (BALF) concentration of the neuropeptide substance P (SP). Exposures to JP-8 were nose-only and for one hour daily. Six groups of Fischer 344 rats were exposed for 7, 28, or 56 days at two JP-8 concentrations (low dose = 469-520 mg/m3/hr, high dose = 814-1263 mg/m3/hr). Exposed groups were matched with longitudinal controls. In response to JP-8 inhalation, exposure animals demonstrated a dose-dependent as well as duration-determined reduction in BALF SP concentration. Both JP-8 concentrations caused significant pathological changes in lower pulmonary structures. We designed this study to elucidate the cause of SP deficits following JP-8 inhalation through correlation with neutral endopeptidase (NEP) concentration taken from paired samples. NEP activity is significantly increased after 28 days of high-dose exposure (HD28) when compared with longitudinal controls and low-dose exposures (7D, 28D, and 56D). A significant inverse relationship between SP and NEP activity is demonstrated through Spearman rank-order correlation (rs = -0.42, n = 52, p < 0.05), suggesting inactivation of SP as the cause of its deficit. Pulmonary airway changes strongly implicate airway epithelium as a primary site of injury. Tachykinin degradation from the peptidase, NEP, plays a role in the process of airway cell injury. This research demonstrates the possible use of NEP and SP lung concentrations as biomarkers of chronic hydrocarbon exposure.