Grantee Research Project Results

Final Report: Responses to Fresh Aerosol in Susceptible Subjects

EPA Grant Number: R832144
Title: Responses to Fresh Aerosol in Susceptible Subjects
Investigators: Kipen, Howard , Lioy, Paul J. , Philipp, Claire , Shindler, Daniel , Laskin, Deborah , Zhang, Junfeng , Ohman-Strickland, Pamela , Laumbach, Robert , Fan, Tina
Institution: Robert Wood Johnson Medical School
EPA Project Officer: Chung, Serena
Project Period: October 1, 2004 through September 30, 2008
Project Amount: $1,521,398
RFA: The Role of Air Pollutants in Cardiovascular Disease (2003) RFA Text | Recipients Lists
Research Category: Particulate Matter , Air Quality and Air Toxics , Air

Objective:

1) Determine if exposure of healthy, young, non-smoking volunteers for 2 hours to freshly generated aerosols will lead to abnormalities in endothelial and platelet function that are independent of pulmonary inflammation. Immediately before and after exposure of subjects to 200 μg/m³ of freshly generated diesel exhaust (DE) or secondary organic aerosol (SOA), and 6 hours post exposure, measurements were made of heart rate variability, endothelial vascular function (brachial artery reactivity and blood nitrite), and platelet activation. To analyze the effects of the aerosols on local pulmonary and systemic inflammation, induced sputum and peripheral blood samples were collected and total leukocyte counts determined.

2) Determine if individuals with genetically increased risk for atherosclerotic cardiovascular disease and endothelial dysfunction exhibit enhanced sensitivity to diesel exhaust or secondary organic aerosol. Recent studies have demonstrated that dysfunction of the vascular endothelium is determined, in part, by a single nucleotide polymorphism (SNP) in the endothelial nitric oxide synthase (eNOS or NOS III) gene. Genotyping was performed to identify subjects with this SNP and matched controls. The effects described in objective 1 are assessed as described.

Summary/Accomplishments (Outputs/Outcomes):

1) Phenotypic Changes in Platelet Activation Using Surface Markers

We have completed flow cytometric analysis of 4 platelet membrane surface markers before and 2 hr after DE and CA exposures in 47 of the 63 subjects. Group means did not show significant changes from DE and/or SOA in cellular mean fluorescence (see 2008-2009 report). However, in an added analysis, when pre-exposure baseline marker levels were regressed against ambient air pollution levels, there were significant decreases in CD42 (GPIb/IX/V complex that manifests decreased surface expression with platelet activation) associated with increased ambient pollution in the 24 hours prior to measurement (Figure 1). This suggests increased platelet activation associated with ambient air pollution, occurring over an acute, but much longer, time span than our diesel exposure measurements, and is consistent with our original hypothesis, albeit at a longer time frame and with a different type of pollutant. It suggests that ambient pollution may be a more potent thrombogenic stimulus than our diesel chamber model of pollution. Other cellular and soluble platelet markers did not show significant responses to ambient pollution.

Figure

Additionally, as included in the 2008-2009 report, because of our relatively null group results for DE-induced platelet activation by flow cytometry, we added two other platelet endpoints to our phenotyping: platelet aggregation on a small subset of more recent subjects and soluble markers of platelet activation on stored plasma samples for the vast majority of subjects.

2) Phenotypic Changes in Platelet Aggregation

In vitro platelet aggregation using epinephrine as an agonist in nine subjects demonstrated a modest increase in platelet aggregation following diesel exhaust exposure with a large decrease following clean air exposure, suggesting that diesel exposure increased aggregation (see 2008-2009 annual report). Logistically, we were unable to perform these add-on analyses on more of our diesel subjects as they had to be immediately transported to Dr. Philipp’s lab 3 miles away. Thus, we were unable to test enough subjects to powerfully reject the null hypothesis of no change. We have applied similar aggregation assays in our daughter study of responses to drastic air pollution changes during the Beijing Olympics, now in the data analysis phase.

3) Phenotypic Changes in Soluble Markers of Platelet Activation

We have also included two additional studies of soluble (rather than cellular) markers of platelet activation in 56 chamber subjects, including the later time point of 6 hr after onset of exposure. sP-Selectin, CD40L and sgpV, showed increases following DE, but neither was statistically significant at either the 2-hour or the 6-hour time points (see 2008-2009 annual report).

4) Overall Assessment for Evidence of Platelet Activation Following Acute Exposure to Diesel Exhaust

Since the initiation of this project, studies from Sweden have been published that have demonstrated increases in platelet aggregation and soluble markers of platelet activation (CD62P) at 2 and 6 hours following acute exposure to diesel exhaust (Lucking et al, 2008). Our study was designed to assess a 2-hour time point for platelet aggregation and a more robust 2- and 6-hour study of soluble markers and found no significant change in either outcome, indicating no increase in coagulation tendency in our system. Our primary method of cellular activation markers of platelets, not previously used in an environmental study and considered the gold standard of platelet activation studies, was null using our diesel inhalation facility. During final analyses, we discovered some previously undetected errors in data entry, and in correcting these, it became evident that there was newly recognized unacceptable variation in platelet results between different flow cytometry technicians over the 3 years of data collection, attributed to flow cytometer gating techniques. In spite of the fact that ICCs for platelet activation were previously demonstrated to be in the acceptable range of 70% (see QA section below), we are presently re-gating all of the 2-hour assays, and this project will be completed in the coming months. We do not anticipate that it will materially change our null results, already confirmed by the methodologically independent and statistically powerful assays of soluble markers from a separate laboratory. We are waiting to see if any impact on the ambient air analyses related to platelet activation changes these significant results shown above.

c) Measurement of Endothelial Function

1. Brachial Artery Ultrasound Scanning (BAUS) Procedure

We have measured endothelial function by flow mediated dilation (FMD%) of the brachial artery. Measurement of brachial artery reactivity was originally done by an ultrasound technician, Ursula Corley, under the direction of Co-Investigator Daniel Shindler, MD, director of the Echocardiography Laboratory at Robert Wood Johnson University Hospital. Because of the departure of Ms. Corley, we identified another technician from RWJUH, Minal Antala. We also initiated collaboration with Dr. Jason Allen of Duke University, who had far more experience with the technique and who optimized our data collection procedures, including use of a “probe holder” to minimize variation between pre and post measurements of artery diameter. We have developed a subcontract with Dr. Allen to perform automated analysis on all of our FMDs, instead of the less reliable manual techniques we originally proposed. The new technician has been trained to collect accurate data under Dr. Allen’s supervision. We collected data on 14 subjects using the new protocol before and after a diesel exhaust and a clean air exposure. The flow-mediated dilation (FMD%) is reported as the percent change in diameter of the brachial artery from baseline. The changes in brachial artery diameter and percent flow-mediated dilation are displayed in the 2008-2009 report. Baseline brachial artery diameter showed a decrease following a 2-hour exposure to both diesel particles and clean air, although it was surprisingly greater for clean air and thus not consistent with previously published reports from the University of Washington. The brachial artery diameter increased as expected following cuff deflation, reaching a peak within 60 seconds of cuff deflation on both exposure days. Percent flow-mediated dilation (FMD%) showed an improvement following a 2-hour exposure to either diesel or clean air particles. Peak FMD% response was observed at 45 seconds following cuff deflation on both exposure days. These data are contrary to what has been hypothesized, as we expected the data to exhibit an acute endothelial dysfunction response and a decrease in FMD% following exposure to diesel exhaust particles. We also failed to observe significant differences between DE and CA exposure days. This suggests an experiment-wise error, although the fact that this was not seen at other centers is perplexing. Alternatively, our data collection for this endpoint may have still been problematic. This technique will require further development before we can use it with confidence.

2. Plasma Nitrite Assessment

Since design of our study, blood nitrite (NO₂) has been reported as an alternative to brachial artery ultrasound scanning (BAUS) and Endo-PAT measurement as a marker of endothelial function (Allen et al, 2005). As a biochemical marker, it is reasonable to examine it in concert with the other, more physiological markers, as it is felt to underlie the physiological vasodilation. We initiated these studies as a consequence of the difficulty we had with the BAUS technique. We have collected blood samples from 49 subjects for measurement of nitrite levels at baseline (pre-exposure) and following a 2-hour exposure to diesel exhaust particles (DE), secondary organic aerosols (SOA), and clean air (CA). Due to restrictions of available staff time required to analyze samples, samples collected on the SOA day are not analyzed. Post minus pre exposure difference for nitrite levels (diff-CA-Nitrite, diff-DE-Nitrite) was calculated for DE and CA days (see 2008-2009 report). These results show, as hypothesized, a greater decrease in nitrite following DE exposure than following exposure to CA. However, there were no statistically significant differences between responses on DE and CA days.

In a quality control review, it was noted by the laboratory that all of the values were uncharacteristically low and ultimately a malfunction was detected in the nitric oxide analyzer (NOA). The analyzer has been repaired and, as of this writing, we have re-run 50% of the nitrite samples, with the remainder to be completed by the end of February 2010. The ranges are as expected; however, we don’t yet know the impact on the overall analysis of diesel effects.

Table 1: Changes in Serum Nitrite Concentration (nM) Following Inhalation of Diesel Exhaust for Two Hours (pending QA correction)

____________________________________________________________________________________________________

Diesel Exhaust (DE) Clean Air (CA)

___________________________________________________________________________________

Pre-Exposure Post-Exposure Diff_DE Pre-Exposure Post-Exposure Diff_CA

N=45 N=45 N=45 N= 45 N=45 N=45

____________________________________________________________________________________________________

Serum Nitrite 166.5 ± 163.8 135.1 ± 114.1 -31.3 ± 123 141.8 ± 123.3 132.8 ± 115.9 -8.9 ± 96.2

____________________________________________________________________________________________________

Values are presented as mean ± SD.

∆ indicates percent pre-exposure to immediate post-exposure absolute change.

3. Measurement of Endothelial function Using the Endo-PAT Tonometer:

This was discussed extensively in the 2008-2009 report. We originally thought that this device would be a simple replacement for BAUS, with which we had very low ICCs. However, in spite of good reliability in the measurements, we had problems ultimately traceable to the tonometer’s unsuitability for use twice within our 2-hour time frame. Apparently, vessels become preconditioned to the ischemic stimulus in a manner that is not a problem for BAUS or blood nitrite measurement. We thus do not consider our pre and post analyses to be reliable.

d) Induced Sputum and Other Pulmonary and Systemic Inflammation Analysis

Sputum induction was performed to assess pulmonary inflammation either at 2 or 6 hours after exposure onset; it is generally not felt to be reliable to induce sputum twice within 24 hours due to the inflammatory effect of the first induction on the second. Consistent with an emerging literature, we did not reliably obtain a valid specimen from our non-asthmatic subjects, a problem with which we were not acquainted prior to this study. 155 sputum samples were collected from 63 subjects. As planned, the minority of samples (28 or 18.1%) was collected at 2 hours following exposure onset and 127 (81.9%) samples were collected at 6 hours following exposure onset. The 2008-2009 report showed detailed QA results for the collected samples. We have used the following literature-based cutoffs for sample adequacy: visible plugs of sputum with total non-red cells greater than 100,000 and less than 25% epithelial cell contamination. We have reported detailed tabular data in the 2008-2009 report, which indicates that 80 of our 155 samples met these eligibility requirements. However, when paired across exposures to DE and CA, we were limited in direct comparison to 22 paired samples (i.e., one subject with a valid specimen after DE and a valid specimen after CA). Comparisons showed greater numbers of WBC (non-squamous cells) following DE, but this was not significant (see 2008-2009 report). Thus, we have equivocal evidence for the presence of our planned gold standard for 6-hour induction of inflammation by the DE.

Table 2 below shows peripheral WBC and RBC counts before and 6 hours after DE exposure. It is clear that no increases following DE, consistent with an inflammatory response, have occurred in this assay from a commercial laboratory.

Table 2

Outcome variable	Pre CA (N=49)	*Post CA (N=48)**	6 hrs post CA (N=47)	Pre DE (N=46)	*PostDE (N=45)**	6 hrs post DE (N=45)
WBC count (Thous/mcL)	6.7 (1.4)	6.6 (1.5)	6.7 (1.5)	6.7 (1.5)	6.7 (1.7)	6.6 (1.6)
RBC count (Thous/mcL)	4.7 (0.4)	4.9 (0.5)	4.7 (0.4)	4.7 (0.4)	4.9 (0.4)	4.7 (0.4)

Cytokines

We did not perform the planned analyses of cytokines in sputum and peripheral blood. This was due to the low number of paired adequate specimens for the sputum. For the peripheral blood, we felt this was a less productive use of financial resources than to pursue novel outcome assays, such as blood nitrite and UPP. In addition, we had access to blood and sputum cytokine assays from a parallel experiment in our facility that was completed approximately 1 year into our study. This study of similar healthy, young subjects was primarily a study of inflammation in response to diesel inhalation (funded by U.S. Department of Defense, N. Fiedler, PI). Exposures were marginally different from ours (300 mg/m³for one hour, producing a cumulative exposure of 300 mg-hours versus our 400 mg-hours). This study measured selected cytokines and total protein 6 hours after DE inhalation. There were no significant changes among 36 pairs of adequate sputum samples for IL-6, IL-8, and TNFα. They also confirmed the present study’s null findings for an increase in sputum cellularity or peripheral WBC count at 6 hours. Plasma IL-6 at both 6 and 24 hours was not significantly changed from baseline, when compared to CA, further documenting the lack of detectable inflammatory response associated with controlled fresh DE exposures in our facility.

e) Genetic Screening for eNOS Polymorphisms

DNA Extraction and Genetic Testing

Buccal swab specimens from 346 volunteers were collected from various campus locations, using the Isohelix DNA cheek swab. These clinical samples were immediately transferred to the EOHSI Bionomics Research and Technology Center (BRTC) for registration and processing. Each sample is assigned a unique barcode for genetic analysis and is entered into the BRTC laboratory information management system (LIMS) for tracking and storage. Within 1 week of collection, each buccal swab sample was treated with digestion buffer, and genomic DNA was extracted using a non-organic solid phase extraction technology to ensure intact genomic DNA samples. All DNA samples go through a rigorous QA/QC procedure following extraction that includes: spectrophotometric analysis (to measure purity), long template PCR (to measure fidelity), and analysis using a standard SNP panel (to ensure no cross contamination and test sample performance) prior to the specific SNP analysis outlined in the proposal. Table 3 displays the distribution of the Glu298Asp SNP stratified by race/ethnicity respectively among all subjects who were screened. In searching for homozygous mutants, we adopted a strategy of screening an excess of white subjects.

Table 3 displays the distribution of the Glu298Asp SNP stratified by race/ethnicity among those subjects who were screened for participation in aerosol exposure

Race	HET	HOM	WT	UNDETERMINED	Total
White	274 (94.8%)	48 (96%)	283 (86.8%)	21 (95.4%)	626 (91.1%)
Black	0 (0%)	1 (2%)	3 (0.9%)	0 (0%)	4 (0.587%)
Asian	13 (4.5%)	1 (2%)	35 (10.7%)	1 (4.5%)	50 (7.2%)
Other	2 (0.6%)	0 (0%)	5 (1.5%)	0 (0%)	7 (1.0%)
Total	289 (42%)	50 (7.2%)	326 (47.4%)	22 (3.2%)	687 (100%)

Note: HET = Heterozygous, HOM = Homozygous, WT = Wild type , UNDETERMINED = No call

Table 4: Distribution of race for Glu298Asp SNP among subjects who have completed participation (N=63)

Race

HET

HOM

Total

White

18 (75%)

14 (100%)

14 (56%)

46 (73%)

Black

0 (0%)

1 (4%)

1 (1.5%)

Asian

5 (20.8%)

0 (0%)

10 (40%)

15 (23.8%)

We have presented selected results above. None of these data can yet be considered fully vetted and complete, in particular the cellular platelet activation and blood nitrite data. However, in general, results from the DE and SOA exposures led to less dramatic and less significant changes than we had hypothesized. We did not clearly demonstrate inflammation, a major hypothesis to explain air pollution effects on CVD, from our system using conventional assays on either peripheral blood or the lung. It remains possible that our exposures were less potent than those in some other systems for particle exposure.

We have been hampered in obtaining BAUS measurement on the intended number of subjects, but in conjunction with the nitrite/nitrate data no strong signal for endothelial dysfunction from the DE condition has emerged. EndoPAT did not prove a reliable alternative to BAUS for our acute studies. Significant platelet activation changes do not appear likely, with our analytical cytometry data supported by the absence of significant changes in the soluble marker data. Once our phenotypic results are confirmed, we will further explore individual genetic susceptibility, although power will be limited as we are restricted to 14 homozygous variant subjects. Ambient air effects on platelet activation are promising but will require confirmation. UPP is our only clearly significant finding and we are exploring its implications, in particular its key role as a regulator of the oxidative stress response. It was statistically significant only when both SOA and DE conditions were combined, although both exposures were associated with decreases in activation compared to clean air in these within-subject analyses..

We have used the experience and results of this study to date as preliminary data for a new NIEHS R01 (ES015864) awarded to Dr. Zhang with Dr. Kipen as Co-I: Response to Drastic Changes in Air Pollution: Reversibility and Susceptibility. We have completed data collection for this project and are now in the analysis phase. A direct extension of the current study (Gene x Environment Interactions after Diesel Exhaust Inhalation in Humans), emphasizing more detailed genotyping of the subjects, as well as additional outcome collection on 35 more subjects, was submitted as an NIH Challenge Grant, but this has not received funding through the main NIH channel.

In collaboration with epidemiologist David Rich we are analyzing additional selected baseline parameters as discussed above (platelet activation, blood counts, EndoPAT) against outdoor ambient air pollution, searching for an association between preceding PM levels and the specified outcomes. This is one way to address the possibility that our diesel exposure is somehow different than those in studies that have reported more robust physiological effects of diesel exhaust.

References:

Allen JD, Cobb FR, Gow AJ. Regional and whole-body markers of nitric oxide production following hyperemic stimuli. Free Radic Biol Med. 2005 May 1;38(9):1164-9.

Lucking AJ, Lundback M, Mills NL, Faratian D, Barath SL, Pourazar J, Cassee FR, Donaldson K, Boon NA, Badimon JJ, Sandstrom T, Blomberg A, Newby DE . Diesel exhaust inhalation increases thrombus formation in man.. Eur Heart J. 2008 Dec;29(24):3043-51

Journal Articles on this Report : 9 Displayed | Download in RIS Format

Publications Views
Other project views:	All 17 publications	10 publications in selected types	All 9 journal articles

Publications
Type	Citation	Project	Document Sources
Journal Article	Conrad L, Rauh V, Hopener L, Acosta L, Perera F, Rundle A, Arteaga-Solis E, Miller R, Perzanowski M. Report of prenatal maternal demoralization and material hardship and infant rhinorrhea and watery eyes. Annals of Allergy Asthma & Immunology 2020;125(4):399.	R832144 (Final) R836154 (2019)	Abstract from PubMed Abstract: Science Direct - Abstract HTML Exit
Journal Article	Kipen HM, Gandhi S, Rich DQ, Ohman-Strickland P, Laumbach R, Fan ZH, Chen L, Laskin DL, Zhang J, Madura K. Acute decreases in proteasome pathway activity after inhalation of fresh diesel exhaust or secondary organic aerosol. Environmental Health Perspectives 2011;119(5):658-663.	R832144 (Final)	Full-text from PubMed Abstract from PubMed Associated PubMed link Full-text: EHP-Full Text HTML Other: EHP-Full Text PDF
Journal Article	Laumbach RJ, Kipen HM. Acute effects of motor vehicle traffic-related air pollution exposures on measures of oxidative stress in human airways. Annals of the New York Academy of Sciences 2010;1203(1):107-112.	R832144 (Final)	Full-text from PubMed Abstract from PubMed Associated PubMed link Abstract: ANYAS-Abstract Exit
Journal Article	Laumbach RJ, Rich DQ, Gandhi S, Amorosa L, Schneider S, Zhang J, Ohman-Strickland P, Gong J, Lelyanov O, Kipen HM. Acute changes in heart rate variability in subjects with diabetes following a highway traffic exposure. Journal of Occupational and Environmental Medicine 2010;52(3):324-331.	R832144 (Final)	Full-text from PubMed Abstract from PubMed Associated PubMed link Abstract: Ovid-Abstract Exit
Journal Article	Laumbach R, Tong J, Zhang L, Ohman-Strickland P, Stern A, Fiedler N, Kipen H, Kelly-McNeil K, Lioy P, Zhang J. Quantification of 1-aminopyrene in human urine after a controlled exposure to diesel exhaust. Journal of Environmental Monitoring 2009;11(1):153-159.	R832144 (Final) R832097 (Final) R832515 aka R832098 (2008)	Full-text from PubMed Abstract from PubMed Associated PubMed link Abstract: RSC-Abstract Exit
Journal Article	Pettit AP, Brooks A, Laumbach R, Fiedler N, Wang Q, Strickland PO, Madura K, Zhang J, Kipen HM. Alteration of peripheral blood monocyte gene expression in humans following diesel exhaust inhalation. Inhalation Toxicology 2012;24(3):172-181.	R832144 (Final)	Full-text from PubMed Abstract from PubMed Associated PubMed link Abstract: Taylor&Francis-Abstract Exit
Journal Article	Rich DQ, Freudenberger RS, Ohman-Strickland P, Cho Y, Kipen HM. Right heart pressure increases after acute increases in ambient particulate concentration. Environmental Health Perspectives 2008;116(9):1167-1171.	R832144 (2007) R832144 (Final)	Full-text from PubMed Abstract from PubMed Associated PubMed link Full-text: EHP-Full Text HTML Other: EHP-Full Text PDF
Journal Article	Sunil VR, Laumbach RJ, Patel KJ, Turpin BJ, Lim H-J, Kipen HM, Laskin JD, Laskin DL. Pulmonary effects of inhaled limonene ozone reaction products in elderly rats. Toxicology and Applied Pharmacology 2007;222(2):211-220.	R832144 (2007) R832144 (Final)	Full-text from PubMed Abstract from PubMed Associated PubMed link Full-text: Science Direct-Full Text HTML Exit Other: Science Direct-Full Text PDF Exit
Journal Article	Sunil VR, Patel KJ, Mainelis G, Turpin BJ, Ridgely S, Laumbach RJ, Kipen HM, Nazarenko Y, Veleeparambil M, Gow AJ, Laskin JD, Laskin DL. Pulmonary effects of inhaled diesel exhaust in aged mice. Toxicology and Applied Pharmacology 2009;241(3):283-293.	R832144 (Final)	Full-text from PubMed Abstract from PubMed Associated PubMed link Full-text: TAP-Full Text HTML Exit Other: TAP-Full Text PDF Exit

Supplemental Keywords:

Diesel exhaust, secondary organic aerosol, endothelial function, platelet activation, cardiovascular disease, vascular nitrite, ubiquitin proteasome pathway (UPP), RFA, Health, Scientific Discipline, Air, HUMAN HEALTH, Susceptibility/Sensitive Population/Genetic Susceptibility, Health Risk Assessment, Risk Assessments, particulate matter, Biology, genetic susceptability, mobile sources, Exposure, Biochemistry, air toxics, inhaled, air quality, environmental hazard exposures, inhaled pollutants, sensitive populations, diesel exhaust, fine particles, lung inflammation, oxidant gas, acute lung injury, human exposure, DEP, engine exhaust, cardiopulmonary responses, particulate exposure, Nitric Oxide Synase, Acute health effects, airway epithelial cells, copollutant exposures, cardiotoxicity, acute exposure, diesel exhaust particulate, chronic health effects, susceptible subpopulations, concentrated particulate matter, air contaminant exposure, toxics, atmospheric particulate matter, heart rate, air pollution, highrisk groups, human susceptibility, airborne urban contaminants, cardiovascular disease, cardiopulmonary, human health risk, ambient particle pollution, cardiopulmonary response, automotive exhaust, diesel engines

Progress and Final Reports:

Original Abstract

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