Grantee Research Project Results
2008 Progress Report: Atherothrombotic Effects of Particulate Matter
EPA Grant Number: EM833367Title: Atherothrombotic Effects of Particulate Matter
Investigators: Bhatnagar, Aruni , Conklin, Daniel
Institution: University of Louisville
EPA Project Officer: Chung, Serena
Project Period: August 1, 2007 through July 31, 2010
Project Period Covered by this Report: August 1, 2007 through July 31,2008
Project Amount: $1,500,000
RFA: Targeted Research Grant (2007) Recipients Lists
Research Category: Targeted Research , Particulate Matter
Objective:
Experiments will be conducted as described in the EPA proposal for performing CAPs exposures in genetically-engineered mice in order to define biological signatures responsive to specific constituents in CAPs (e.g., aldehydes, metals).Progress Summary:
Description of Inhalation Facility at University of Louisville
In preparation for EPA-sponsored project, we began construction of a ~1,600 ft2 inhalation facility, which contains a barrier style entry way, a walk-through autoclave, an animal housing room (capacity ~4,000 mice), a surgical procedures room, and an ~400 ft2 inhalation room (Figs. 1-3). All rooms are fully equipped and operational. The animal room was certified as pathogen-free in March 2008 after 3 months of sentinel mice testing, and we have been housing and ordering mice into the facility since then.
The inhalation facility operates as a barrier facility with card-code access only since April 2008. The animal facility is maintained by the University animal resources staff with staff access only to the animal room and autoclave area. We have continued to operate sero-negative since then, and we now are housing ~50% of our breeding colonies in this facility. The procedures room has been prepared for performing survival surgery including surgical implantation of telemetric devices for recording blood pressure and electrocardiogram in conscious mice during exposures as well as in home cages after exposures. The telemetry system has 2 data matrices, 8 receivers, 8 blood pressure transmitters, and 8 ECG transmitters as well as all necessary software for data collection and analysis (DSI, St. Paul, MN). We have received surgical training from DSI for implantation of ECG and blood pressure (with cannulation at left carotid artery and abdominal aorta sites) transmitters. Blood pressure and ECG will be monitored during PM exposures as well as in our ongoing acrolein exposures.
The inhalation room has slight negative pressure relative to the procedures room and to the outside, and has equipment for performing acrolein and chlorine gas inhalation exposures, as well as, a custom-designed versatile aerosol concentration and enrichment system (VACES) modeled after a system currently used by Dr. Lung-Chi Chen (New York University; Fig. 2AB, Fig. 3). We visited Dr. Chen’s laboratory and received 3-days of training from Dr. Chen at NYU in January 2008, and we are nearly completed construction of the UofL VACES. In contrast to other systems, our VACES features 4 independent lines: 2 lines for filtering air and 2 for concentrating particles. For filtering and concentrating ambient particles we have a fine PM (PM2.5) using a cyclone impactor and an ultrafine PM (UFP) line that uses a custom-designed UFP cut-plate (Dr. C. Sioutas, UCLA) made for our collaborator, Dr. Q-W. Zhang (School of Public Health, Univ. of Louisville). In addition to these two devoted lines to PM, we have 2 HEPAfiltered ambient air lines for air control exposures.
In addition, we have the option of directing either the fine PM or UFP line to a biosampler line for bulk collection of these particles for comprehensive physico-chemical particle analyses or for use of particles in in-vitro studies. With these 4 exposure lines, we can simultaneously expose up to 4 individual exposure chambers; each chamber houses up to 16 mice per chamber. Because these stainless chambers have plexiglass tops and perforated stainless steel dividers, we can record blood pressure or ECG from 4 mice per chamber. Although we have had some logistical and infrastructure delays during development of the UofL VACES, we believe the infrastructure, equipment and personnel are in place to initiate our proposed studies of the atherothrombotic effects of Louisville air PM in mice. Thus, we are progressing in the direction of the grant to accomplish the specific proposed experiments of the grant at this time.
Progress in Acrolein, Cigarette Smoke and Diesel Exposures:
Because our basic premise states that environmental aldehydes contribute to the cardiovascular toxicity of air, water, and food pollutants – and especially as a component of fine and ultrafine PM, we have performed dose- and time-dependent acrolein inhalation exposures in mice. Exposures have lasted from 1-5 days in C57BL/6 mice (at UofL) and ~10 weeks in apoE-/- mice at NYU (Dr. LC Chen, collaborator) using levels of 0.1-5 ppm (Fig. 4). We have assessed dyslipidemia, endothelial dysfunction, thrombosis and atherosclerosis in these mice. Collectively, data from these studies indicate that acrolein induces prothrombotic and proatherosclerotic changes in dose dependent manner and these data are being readied for publication.
Similarly, we have collaborated over the past years on 2 different projects that bear upon cardiovascular effects of environmental pollutant exposure and we believe these collaborations have helped establish our ability to evaluate critically the appropriate biological endpoints for pollutant exposure in terms of increased cardiovascular disease risk. One study is a collaborative effort with Dr. Judy Zelikoff, NYU, to evaluate the effects of prenatal mainstream cigarette smoke exposure on adult lipid profile in mice and the potential synergistic effects of post-natal high fat diet. These studies revealed long-lasting, epigenetic changes in the smoke-exposed mice following an acute high fat diet challenge in adult life. These data support the hypothesis that fetal exposures and fetal programming are important contributors to increased cardiovascular disease risk in adults. Our lab was instrumental in performing lipids analyses in these studies and establishing these as appropriate endpoints in normolipidemic C57BL/6 mice. This study is submitted and under review at EHP.
Additionally, we have collaborated with Dr. Matthew Campen, Lovelace Respiratory Research Institute, in a NCER-funded study of atherosclerotic effects of diesel emissions and the specific role of particles. We have evaluated both lipids and atherosclerosis in these mice and results of this study are being readied for publication. Again, results from this study support the contention that particles contribute to some, but not all, of the detrimental cardiovascular effects of
diesel exposure in mice. Collectively, these studies serve as evidence of our commitment to understanding the cardiovascular effects of airborne pollutants and have honed our technical skills for critical evaluation of the effects of exposures to fine and ultrafine PM in Louisville air in mice. Therefore, we are poised and excited to conduct our studies as proposed. With our collaborator, Dr. Russell Prough, Dept. Biochemistry and Mol. Biol. at Univ. of Louisville, we have developed a genetic signature for C57BL/6 mice and the Nrf2-null mice by comparing the effects of the oxidant butylated hydroxyanisole (BHA) exposure with the effects stimulated by acrolein exposure (Table 1).
This study will serve as a test-case for the utility of our basic approach, which posits that specific chemical exposure produces a specific biological signature in the cardiovascular system that is discernable from other chemical stimuli because it provokes a select set of adaptive genes that are regulated by specific transcription factors. A small subset of data comparing the effects of BHA and the effects of acrolein exposure on hepatic cytochromes P450 (CYP) gene mRNAs indicates that while there are gene effects that are shared in common between the two exposures, the patterns are also distinct indicating that other transcription factors in addition to Nrf2 regulate
CYP gene expression (Table 1). These patterns will be compared in our genetic model mice, Nrf2- and MFT1-null mice, as well.
Results to Date:
Although we have not performed CAPs exposures to date, we expect to perform our first acute exposures in the near future of year 2. In year 1, we have performed numerous acute acrolein inhalation exposures as indicated above, and we have established the protocols for animal handling, tracking, post-exposure monitoring and developed a biological “signature” of acrolein inhalation exposure. Because our basic premise states that environmental aldehydes contribute to the cardiovascular toxicity of air, water, and food pollutants – and especially as a component of fine and ultrafine PM, we have performed dose- and time-dependent acrolein inhalation exposures in mice. Exposures have lasted from 1-5 days in C57BL/6 mice (UofL Inhalation Facility) and ~10 weeks in apoE-/- mice at NYU (Dr. LC Chen, collaborator) using levels of 0.1-5 ppm (Fig. 4). We have assessed functional endpoints such as endothelial dysfunction, thrombosis and atherosclerosis in these mice. Collectively, data from these studies indicate that acrolein induces prothrombotic and proatherosclerotic changes in dose dependent manner and these are being readied for publication. Additionally, in recent experiments, we have developed assays to assess the number and function of bone marrow and circulating endothelial progenitor cells, known as EPCs using flow-activated cell sorting (FACS) techniques. Because of these new findings, we are developing new R01 applications to investigate effects of environmental exposures on EPCs and platelets. As these data will serve as preliminary data for grants, we have not published or presented these data as yet.
Future Activities:
We plan to carry out our experiments as planned in our initial proposal. The facility and VACES construction is complete and we are now moving to the next phase of the project in year 2. This phase is to perform acute and chronic exposures in mice using HEPA-filtered and concentrated ambient particle air (CAPs; fine and ultrafine) using a newly constructed VACES. Genetically-engineered mice will be used to interrogate the role of specific particulate matter constituents (e.g., metals, aldehydes) by comparing the biological responses to CAP exposure, which serve as biological “signatures” of chemical exposure.
Journal Articles on this Report : 4 Displayed | Download in RIS Format
Other project views: | All 25 publications | 18 publications in selected types | All 16 journal articles |
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Conklin DJ, Haberzettl P, Prough RA, Bhatnagar A. Glutathione S-transferase P protects against endothelial dysfunction induced by exposure to tobacco smoke. American Journal of Physiology Heart and Circulatory Physiology 2009;296(5):H1586-H1597. |
EM833367 (2008) EM833367 (2009) EM833367 (Final) |
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Conklin DJ, Barski OA, Lesgards JF, Juvan P, Rezen T, Rozman D, Prough RA, Vladykovskaya E, Liu S, Srivastava S, Bhatnagar A. Acrolein consumption induces systemic dyslipidemia and lipoprotein modification. Toxicology and Applied Pharmacology 2010;243(1):1-12. |
EM833367 (2008) EM833367 (Final) |
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Haberzettl P, Vladykovskaya E, Srivastava S, Bhatnagar A. Role of endoplasmic reticulum stress in acrolein-induced endothelial activation. Toxicology and Applied Pharmacology 2009;234(1):14-24. |
EM833367 (2008) EM833367 (2009) EM833367 (Final) |
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O'Toole TE, Zheng YT, Hellmann J, Conklin DJ, Barski O, Bhatnagar A. Acrolein activates matrix metalloproteinases by increasing reactive oxygen species in macrophages. Toxicology and Applied Pharmacology 2009;236(2):194-201. |
EM833367 (2008) EM833367 (2009) EM833367 (Final) |
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Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.