Lee S. Newman

Human health risk assessment of air emissions from development of unconventional natural gas resources

BACKGROUND Technological advances (e.g. directional drilling, hydraulic fracturing), have led to increases in unconventional natural gas development (NGD), raising questions about health impacts. OBJECTIVES We estimated health risks for exposures to air emissions from a NGD project in Garfield County, Colorado with the objective of supporting risk prevention recommendations in a health impact assessment (HIA). METHODS We used EPA guidance to estimate chronic and subchronic non-cancer hazard indices and cancer risks from exposure to hydrocarbons for two populations: (1) residents living >½ mile from wells and (2) residents living ≤ ½ mile from wells. RESULTS Residents living ≤ ½ mile from wells are at greater risk for health effects from NGD than are residents living >½ mile from wells. Subchronic exposures to air pollutants during well completion activities present the greatest potential for health effects. The subchronic non-cancer hazard index (HI) of 5 for residents ≤ ½ mile from wells was driven primarily by exposure to trimethylbenzenes, xylenes, and aliphatic hydrocarbons. Chronic HIs were 1 and 0.4. for residents ≤ ½ mile from wells and >½ mile from wells, respectively. Cumulative cancer risks were 10 in a million and 6 in a million for residents living ≤ ½ mile and >½ mile from wells, respectively, with benzene as the major contributor to the risk. CONCLUSIONS Risk assessment can be used in HIAs to direct health risk prevention strategies. Risk management approaches should focus on reducing exposures to emissions during well completions. These preliminary results indicate that health effects resulting from air emissions during unconventional NGD warrant further study. Prospective studies should focus on health effects associated with air pollution.

Beryllium sensitivity is linked to HLA-DP genotype.

Chronic beryllium disease (CBD) appears to arise from a combination of both exposure and genetic risk factors. A distinguishing feature of CBD is beryllium hypersensitivity, which can be measured in vitro by a lymphocyte proliferation test. The objective of this study was to determine whether certain allelic variations of the HLA-DPB1 gene, which had been observed previously in CBD, could be found in a group of individuals having beryllium hypersensitivity, but no symptoms of CBD. A flow cytometry-based Lymphocyte Proliferation Test combined with immunophenotyping (Immuno-LPT) was used to detect CD4+ and CD8+ T cell proliferation in response to in vitro stimulation with beryllium. The HLA-DPB1 haplotypes of the same individuals were determined by automated DNA sequencing. Twenty-two out of 25 beryllium-sensitive, non-CBD individuals were found to be carriers of the HLA-DPB1 gene having a substitution of a glutamic acid at position 69 in Exon 2 (Glu69), and a significantly high percentage (24%) were Glu69 homozygotes. Most of the CD4+ responders on the Immuno-LPT (10/14) carried rare, non-*0201 Glu69 DPB1 alleles; while most of the non-CD4+ responders (9/11) were common Glu69 carriers (*0201 or *0202) or non-Glu69 individuals (non-Glu69/non-Glu69). This is the first direct evidence that HLA-DP genotype is linked to a phenotypic response that occurs in beryllium sensitization in the absence of clinical CBD.

Genetic and environmental modulation of chronic obstructive pulmonary disease

Chronic Obstructive Pulmonary Disease (COPD) is a leading cause of morbidity and mortality throughout a large part of the western world. Although personal tobacco use has been implicated in a large number of these cases, it is also true that only a fraction of smokers ever develop respiratory problems. Therefore, the question of host susceptibility and other environmental factors should be considered. This paper will briefly review evidence for host susceptibility to COPD, review evidence for additional environmental risk factors for the development of COPD, and give an example of environmental interactions with a known genetic risk factor that further increase the risk of COPD.

Beryllium Skin Patch Testing to Analyze T Cell Stimulation and Granulomatous Inflammation in the Lung

Chronic beryllium disease (CBD) is characterized by granulomatous inflammation and the accumulation of CD4+ T cells in the lung. Patch testing of CBD patients with beryllium sulfate results in granulomatous inflammation in the skin. We investigated whether the T cell clonal populations present in the lung of CBD patients would also be present in the involved skin of a positive beryllium patch test and thus mirror the granulomatous process in the lung. CBD patients with clonal TCR expansions in bronchoalveolar lavage (BAL) were selected for study. All three CBD patients studied had a positive response to beryllium sulfate application and a negative patch test to normal saline. Immunohistochemistry showed extensive infiltration with CD4+ T cells and few, if any, CD8+ T cells both at 3 days and at later times when granulomas were apparent. T cell infiltration early after skin testing appeared to be nonspecific with the TCR repertoire of infiltrating T cells being distinct from that present in BAL. At later times when granulomas were present, T cell clones in skin overlapped with those in BAL in all patients tested. Total TCR matches in skin and BAL were as high as 40% in selected Vβ T cell subsets. Studies of peripheral blood T cells before and after patch testing provided evidence for mobilization of large numbers of pathogenic beryllium-reactive T cells into the circulating pool. These studies using skin patch testing provide new insight into the dynamics of T cell influx and mobilization during granulomatous inflammation.

Nontuberculous Mycobacteria in Aerosol Droplets and Bulk Water Samples from Therapy Pools and Hot Tubs

Hot tub exposure has been causally associated with a steroid-responsive, granulomatous lung disease featuring nontuberculous mycobacterial (NTM) growth in both clinical and environmental samples. Little is known regarding prevalence of and risk factors for NTM-contamination and associated illness in these settings. In this study, the frequency of NTM growth and aerosolization in 18 public hot tubs and warm water therapy pools and the factors associated with mycobacterial growth were analyzed. Each site was characterized by water chemistry analysis; a questionnaire on maintenance, disinfection, and water quality; and air and water sampling for quantitative NTM culture. NTM were detected in air or water from 13/18 (72%) sites; a strong correlation was found between the maximum air and water NTM concentrations (rho 0.49, p = 0.04). Use of halogen (chlorine or bromine) disinfection was associated with significantly lower air and water concentrations of NTM compared with disinfection using ultraviolet light and hydrogen peroxide (p = 0.01–0.04). Higher water turnover rates were also associated with lower air and water NTM concentrations (p = 0.02–0.03). These findings suggest that NTM are frequently detectable in the air and water of spas and therapy pools and that particular maintenance and disinfection approaches affect NTM bioaerosol concentrations in these settings.

TGF-β1 Variants in Chronic Beryllium Disease and Sarcoidosis

Evidence suggests a genetic predisposition to chronic beryllium disease (CBD) and sarcoidosis, which are clinically and pathologically similar granulomatous lung diseases. TGF-β1, a cytokine involved in mediating the fibrotic/Th1 response, has several genetic variants which might predispose individuals to these lung diseases. We examined whether certain TGF-β1 variants and haplotypes are found at higher rates in CBD and sarcoidosis cases compared with controls and are associated with disease severity indicators for both diseases. Using DNA from sarcoidosis cases/controls from A Case Control Etiologic Study of Sarcoidosis Group (ACCESS) and CBD cases/controls, TGF-β1 variants were analyzed by sequence-specific primer PCR. No significant differences were found between cases and controls for either disease in the TGF-β1 variants or haplotypes. The −509C and codon 10T were significantly associated with disease severity indicators in both CBD and sarcoidosis. Haplotypes that included the −509C and codon 10T were also associated with more severe disease, whereas one or more copies of the haplotype containing the −509T and codon 10C was protective against severe disease for both sarcoidosis and CBD. These studies suggest that the −509C and codon 10T, implicated in lower levels of TGF-β1 protein production, are shared susceptibility factors associated with more severe granulomatous disease in sarcoidosis and CBD. This association may be due to lack of down-regulation by TGF-β1, although future studies will be needed to correlate TGF-β1 protein levels with known TGF-β1 genotypes and assess whether there is a shared mechanisms for TGF-β1 in these two granulomatous diseases.

Chronic beryllium disease: a model interaction between innate and acquired immunity.

Beryllium (Be) is a lightweight and durable metal useful to a variety of manufacturing processes. With the use of Be in industrial settings, a number of health effects were noted including acute pneumonitis, sensitization to Be, interstitial lung disease and dermatological disease. Interstitial mononuclear cell inflammation and granuloma formation are the primary processes that occur in the lungs of Be-exposed workers, resulting in chronic beryllium disease (CBD). Recent studies have begun to describe the role of Be in the pathogenesis of CBD. These studies reveal that the hosts response to Be involves components of the innate immune system or inflammatory responses. Inflammatory responses to Be can establish a state of acquired, Be antigen-specific, cell-mediated immunity. Despite triggering both the innate and acquired immune responses, Be is not eliminated from the host. Rather, it establishes pathways leading to chronic granulomatous inflammation. We will examine recent studies describing the hosts cellular and molecular responses to Be, responses that promote granuloma formation.

Occupational exposures in the oil and gas extraction industry: State of the science and research recommendations

The oil and gas extraction industry is rapidly growing due to horizontal drilling and high volume hydraulic fracturing (HVHF). This growth has provided new jobs and economic stimulus. The industry occupational fatality rate is 2.5 times higher than the construction industry and 7 times higher than general industry; however injury rates are lower than the construction industry, suggesting injuries are not being reported. Some workers are exposed to crystalline silica at hazardous levels, above occupational health standards. Other hazards (particulate, benzene, noise, radiation) exist. In this article, we review occupational fatality and injury rate data; discuss research looking at root causes of fatal injuries and hazardous exposures; review interventions aimed at improving occupational health and safety; and discuss information gaps and areas of needed research. We also describe Wyoming efforts to improve occupational safety in this industry, as a case example.

Beryllium-stimulated production of tumor necrosis factor-α by a mouse hybrid macrophage cell line

Chronic beryllium disease (CBD) results from exposure to the light-weight metal beryllium (Be). In vitro stimulation of bronchoalveolar lavage cells from CBD subjects causes the production of high levels of TNF-alpha, IFN-gamma and IL-6. We tested the hypothesis that Be-stimulation might induce the production of TNF-alpha by macrophage cell lines. We observed that H36.12j cells (12j), a mouse hybrid macrophage cell line, but not other mouse and human macrophage cell lines, produced TNF-alpha upon Be-stimulation. The response was maximal at 100 microM BeSO4 and did not occur when 12j cells were stimulated with either aluminum sulfate or cobalt sulfate. Beryllium-stimulated the production of 725+/-25 pg/ml (mean +/- SEM) TNF-alpha protein by 12j cells as measured by ELISA of culture supernatants after 24 h. As measured by RT-PCR, Be-stimulated 12j cell TNF-alpha protein production was accompanied by an increased intracellular TNF-alpha mRNA at 3 and 24 h. The addition of 10U or 100U of rMu-IFN-gamma to Be-stimulated 12j cells further increased TNF-alpha production 1.5-4 fold (1.6+/-0.1 ng/ml) respectively. Bacterial lipopolysaccharide (LPS, 1 microg/ml) stimulated production of TNF-alpha in 12j culture supernatants after 6 h (515+/-151 pg/ml). This early versus late TNF-alpha production suggests that LPS and Be both stimulate 12j cell TNF-alpha synthesis, but through different pathways. We report for the first time, the direct effects of Be stimulation on the ability of 12j cells to produce TNF-alpha. The 12j cell line, contrasted with other macrophage hybrids that do not respond to Be-stimulation, may provide a useful tool to evaluate the mechanisms by which Be stimulates macrophage cytokine production, and by which T cell derived IFN-gamma amplifies TNF-alpha production in granulomatous diseases.

Beryllium-stimulated in vitro migration of peripheral blood lymphocytes

Inhalation of beryllium (Be) induces both inflammatory and metal antigen-specific immune responses in the lungs characterized by mononuclear cell infiltration and granuloma formation (chronic beryllium disease, CBD). We tested the hypothesis that Be-salts might increase the in vitro migration of peripheral blood mononuclear cells (PBMC). PBMC are mixed cells, consisting of lymphocytes and monocytes. We compared their responses to populations of both purified blood lymphocytes, and purified blood monocytes. Purified blood monocytes and lymphocytes, isolated by Percoll gradients and centrifugal elutriation from normal human subjects (n = 6), were exposed to graded concentrations (0.01 to 100 microM) of BeSO4 or to the control metal-salt Al2(SO4)3. Migratory responses of stimulated PBMC were measured in Boyden Chambers. As controls, PBMC mixed cells or purified lymphocytes or purified monocytes were unstimulated or stimulated with a positive chemoattractant, Zymosan-A treated pooled, normal human serum (ZAS). The migration index (MI) was defined as the distance (micrometers) that cells migrated through a 5 micron filter. The MI for unstimulated PBMC mixed cells was 75+/-4 whereas the MI for ZAS-stimulated PBMC mixed cells was 124+/-4 (P < or = 0.05, Tukey-Kramer). The MI for BeSO4 -stimulated (100 microM) PBMC mixed cells was 136+/-4. The observed increase in the BeSO4-stimulated PBMC mixed cell migration was significant down to 0.1 microM BeSO4. BeSO4, BeCl2 and BeF2, tested at 100 and 10 microM, were equally effective at inducing PBMC mixed cell migration. Equimolar concentrations of Al2(SO4)3 were not as effective at inducing PBMC mixed cell migration, MI < 100 at 100 microM, and did not induce PBMC mixed cell migration at concentrations below 1 microM. The migration of purified monocytes through filters was not increased in response to either BeSO4 or Al2(SO4)3 compared to controls, but did respond to ZAS (MI = 100+/-4). Purified lymphocytes migrated in response to stimulation with all concentrations of BeSO4 tested (100 microM MI = 133+/-9), and Al2(SO4)3 (100 microM MI = 85+/-8). There were no significant differences in the MI for PBMC mixed cells or for purified lymphocytes at the concentrations of BeSO4 tested. Our data show that Be directly induces the in vitro migration of PBMC mixed cells and purified blood lymphocytes, and not purified blood monocytes, across a broad range of Be concentrations. This induction of migration was independent of the molecular form of the Be-salt. Inhaled Be, by promoting lymphocyte emigration to the lung, may create a microenvironment that favors a Be-antigen-specific T-lymphocyte response, chronic inflammation, and CBD.