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Final Report: Biomarker Core
EPA Grant Number: R827355C010Subproject: this is subproject number 010 , established and managed by the Center Director under grant R827355
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Airborne PM - Northwest Research Center for Particulate Air Pollution and Health
Center Director: Koenig, Jane Q.
Title: Biomarker Core
Investigators: Kalman, Dave , Dills, Russell , Simpson, Chris
Institution: University of Washington
EPA Project Officer: Stacey Katz/Gail Robarge,
Project Period: June 1, 1999 through May 31, 2004 (Extended to May 31, 2006)
RFA: Airborne Particulate Matter (PM) Centers (1999)
Research Category: Particulate Matter
Description:
Objective:- Validate the use of organic tracers to apportion woodsmoke contribution to ambient PM and to determine environmental levels of and personal exposures to biomass generated PM.
- Validate a urinary biomarker of exposure to woodsmoke.
- Develop the DTT assay as a measure of the oxidative potential of PM samples.
1) Validation of the use of organic tracers to apportion woodsmoke contribution to ambient PM and to determine environmental levels of and personal exposures to biomass generated PM.
We have analyzed >200 personal filters from the Seattle panel study for the woodsmoke tracer levoglucosan. These data were used to estimate personal exposures to woodsmoke. We also have measured levoglucosan levels in ~150 personal and central site filters from the study of health effects of exposure to agricultural burning smoke in Pullman, WA. These data were used to define episodes of field burning smoke impacts in Pullman and to assess personal exposures to biomass smoke. Methoxyphenol levels were measured in ~100 personal and central site filters from this study also. Filter samples containing various types of PM collected in Thailand were analyzed for levoglucosan and methoxyphenols. These data are being used to construct source profiles for rice burning and urban aerosol in Thailand, and will be used in source apportionment studies (in collaborative with a Fogarty Grant at UW).
2) Validatation of a urinary biomarker of exposure to woodsmoke.
We have addressed this aim by examining methoxyphenol exposure and excretion in a variety of populations with different levels of exposure to biomass smoke. Our initial analyses of data from these cohorts is summarized in Figure 2. In the figure, woodsmoke exposure in these populations is represented by airborne levoglucosan concentrations (personal monitoring for the farmer, campfire and Seattle studies, indoor monitoring for the Guatemala study). We expect that levoglucosan is the most appropriate marker of external exposure to woodsmoke (or woodsmoke levels in ambient air samples) because in comparison to the methoxyphenols it is more abundant (therefore more readily measured in low volume samples), it appears to have greater chemical stability, and it is unaffected by issues of particle phase-vapor phase partitioning. Urinary methoxyphenol excretion in figure 2 is represented by unadjusted urinary syringol concentrations.
Figure 2: Woodsmoke exposure and urinary syringol concentrations in cohorts that represent a range of woodsmoke exposures. (Asterisk indicates woodsmoke exposure estimated using regional average values).
Two key observations are apparent from observation of the data in Figure 2. Firstly, a significant increase in urinary methoxyphenol excretion is only observed after high woodsmoke exposures (i.e., Guatemala study, campfire study), on the order of 10 μg/m3 levoglucosan. Secondly, background methoxyphenol concentrations in urine are relatively high and variable, and they appear to respond to efforts to suppress methoxyphenol intake in the diet. For example, Seattle residents on an unrestricted diet had urinary syringol concentrations of ~0.2-0.6 μg/mL. Participants in the managed exposure study who were asked to refrain from consuming foods or beverages that contained woodsmoke flavorings had urinary syringol concentrations of ~0.008 μg/mL, and a subject on a severely restricted diet (rice and boiled vegetables) had urinary syringol concentrations of ~0.003 μg/mL. Thus the prerequisites for the successful use of urinary methoxyphenols as biomarkers of exposure to woodsmoke are either that the woodsmoke exposure are substantial, or that dietary consumption of methoxyphenols is suppressed such that lower levels of woodsmoke exposure may be reflected in urinary methoxyphenol excretion.
A managed exposure study was undertaken in which nine subjects were exposed to woodsmoke (mixture of hard and softwoods) inside a yurt for a two hour period., Exposure data from this study is illustrated in Figures 3 and 4. A good correlation is evident between the two measures of smoke exposure. This figure also illustrates a high level of short term variability in the smoke levels: peak PM2.5 concentrations were >30 μg/m3, which is more than 10-fold greater than the time-weighed average PM2.5 concentrations measured with the same instrument (1576 μg/m3).
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Figure 3: Woodsmoke exposures from open fire |
Figure 4: Continuous PM and CO exposures from open fire (CO data offset for clarity) |
Personal PM2.5 exposures experienced by the subjects during the managed exposure study are illustrated in figure 3. The average PM2.5 exposure was 1515 μg/m3, and there was a 3.5 fold range between the subject with the lowest exposure (840 μg/m3) and the subject with the highest exposure (2997 μg/m3). Fine PM exposures of this magnitude are high in comparison to ambient air quality guidelines (PM2.5 NAAQS = 65 μg/m3 for 24 hours), but are within the range of exposures experienced by wildland firefighters (e.g. mean cross shift exposures ~500 – 630 μg/m3, maximum exposures ~2,930 – 6,900 μg/m3 (Reinhardt,2000a; Reinhardt,2000b), communities impacted by wildland fire smoke (e.g. Hoopa Indian Reservation 600 mg/m3 PM10 (24 hr average in 1999) (Mott,2002), Hamilton, MN 350 μg/m3 PM2.5 (24 hr average in 2000), and indoor air exposures in less developed countries where biomass fuels are widely used (e.g. India 700-800 μg/m3 PM10 (Smith,2000)).
Figure 5: Timecourse of urinary excretion of syringol for nine subjects exposed to woodsmoke
Measurement of 22 methoxyphenols, levoglucosan, and polynuclear hydrocarbons was performed by GC/MS assays for personal filter samples and urine samples collected for 3-days centered on the exposure. Most urinary methoxyphenols had appreciable pre-exposure levels; for 7 methoxyphenols (propylquaiacol, cis- & trans-isoeugenol, syringol, methylsyringol, ethylsyringol and propylsyringol) consistently among subjects, peak urinary concentrations occurred after the woodsmoke exposure. Eight subjects had peak urinary elimination of methoxyphenols within 10 hours (t1/2 3-5 hr); whereas one subject had delayed elimination. The timecourse of excretion is illustrated for syringol in figure 5. Several metrics for urinary excretion were evaluated. Concentration was greatly affected by urine volumes. Excretion rate and concentrations normalized by creatinine gave a much clearer signal and were found to be essentially equivalent in predictive ability. 12-hr average creatinine-normalized concentrations of each of the 7 methoxyphenols gave a Pearson correlation ≥ 0.8 with the air concentration of the corresponding methoxyphenol. The sum of concentrations for the7 methoxyphenols versus levoglucosan on personal filters gave a regression coefficient of 0.78. The same sum versus the personal PM2.5 gave a regression coefficient of 0.77. Consideration of the intercept of this regression would suggest that the threshold for detection of an acute exposure event would be approximately 400 μg/m3 wood smoke. The signal to noise (12-hr post exposure average/pre-exposure average) ranged from 3–20 for the 7 methoxyphenols. Analysis of multiple compounds provided assurance that elevations were not artifactual due to food or other substances.
3) Develop the dithiothrietol (DTT) assay as a measure of the oxidative potential of PM samples.
Work has continued to validate the dithiothrietol assay for oxidative potential of PM (DTT assay) in our laboratory. This assay has been applied to PM samples from various sources including woodsmoke, diesel SRM and fresh diesel exhaust from our exposure facility. Of all these samples, woodsmoke showed the highest response.
We have shown previously that specific collected aerosol samples possess innate chemical reactivity (Simpson,2004). PM2.5 from Seattle, WA collected on Teflon filters catalyzed oxidation and nitration of methoxyphenols during ultrasonication in ethylacetate. The chemical transformation products were identified by GC/MS. Two standard reference materials (SRM 2975, diesel exhaust particulate; SRM 1649a, urban dust) also caused breakdown of methoxyphenols under these conditions. The chemical breakdown of the methoxyphenols can be prevented through the addition of triethylamine to the extraction solvent. These observations demonstrate that air particulate samples possess the ability to promote oxidative transformation of organic chemicals under relatively mild conditions. Given the significance of oxidative stress as a mechanism by which PM is proposed to cause toxicity, measures of the oxidative capacity of air particulate samples are biologically relevant PM characteristics for study.
We have used the DTT assay to examine various particulate matter preparations for oxidant activity. In these assays, we extract the particulate matter with methanol and then filter the extract. Extracts of diesel particulate SRM, and wood smoke particulate have consistently shown high activity in the assay, while house dust and recently formed diesel particulate from a modern engine show virtually no activity (Figure 6).
Figure 6: Oxidant capacity of PM samples. WS = wood smoke particulate PQ = phenanthroquninone (positive control), SRM = diesel SRM 2975
We are in the process of identifying the active oxidant species in the diesel particulate SRM and wood smoke particulate. We applied classic pH-based separation techniques to the extracts of these particulates. Analysis of the acidic, basic, neutral, and phenolic fractions demonstrated that >50% of the activity resided in the phenolic and acidic fractions. DTT activity was generally ( >80%) conserved during the fractionation process.
Each of the four pH-fractions derived from wood smoke particulate was further sub-fractionated. Five fractions of approximately equal volume were generated by reversed-phase HPLC using a 5-100% gradient of water to methanol. Activity was found to reside in the fractions containing phenols, many of which were identified as guaiacols and syringols which are frequently used as tracers for wood smoke. However, we found that a synthetic mixture containing these known phenols did not give an equal response in the DTT assay. Other potentially bioactive components the we identified by GC/MS in the ‘phenolic’ HPLC fractions include alkyl and hydroxy substituted benzoic and acetic acids, benzene diols, naphthols, methyoxy-naphthalenes, hydroxybenzopyrans, hydroxybenzils, benzofuranones, mono and di-hydroxybenzaldehydes, and adipate esters.
We characterized the biological activity in PM extracts by using cell culture cytotoxicity assays. The biological activity of the organic extracts of wood smoke particulate and diesel particulate reference material was examined using an in vitro urothelial cell cytotoxicity assay (Figure 7). As was observed in the DTT assay (above) the wood smoke extracts showed greater toxicity than the diesel SRM extract. Much of the biological activity of the wood smoke and diesel exhaust extracts extract was accounted for by the K2CO3 (acidic/phenolic) fraction, as was also observed in the DTT assay. Furthermore, our preliminary data provides support for our underlying hypotheses: Chemical reactivity (as assessed by the DTT assay) was substantially preserved following chemical fractionation of woodsmoke and diesel exhaust particulates, and was enriched in the phenolic/acidic fractions (Table 5). Chemical fractions with the highest DTT response were also the most cytotoxic in the in vitro bioassay Figure 7).
Journal Articles on this Report: 12 Displayed | Download in RIS Format
| Other subproject views: | All 19 publications | 12 publications in selected types | All 12 journal articles |
| Other center views: | All 191 publications | 97 publications in selected types | All 94 journal articles |
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Clark M, Paulsen M, Smith KR, Canuz E, Simpson CD. Urinary methoxyphenol biomarkers and woodsmoke exposure: comparisons in rural Guatemala with personal CO and kitchen CO, levoglucosan, and PM2.5. Environmental Science & Technology 2007;41(10):3481-3487. |
R827355 (Final) R827355C010 (Final) |
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Dhammapala R, Claiborn C, Simpson C, Jiminez J. Emission factors from wheat and Kentucky bluegrass stubble burning: comparison of field and simulated burn experiments. Atmospheric Environment 2007;41(7):1512-1520. |
R827355 (Final) R827355C010 (Final) |
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Dhammapala R, Claiborn C, Jimenez J, Corkill J, Gullet B, Simpson C, Paulsen M. Emission factors of PAHs, methoxyphenols, levoglucosan, elemental carbon, and organic carbon from simulated wheat and Kentucky bluegrass stubble burns. Atmospheric Environment 2007;41(12):2660-2669. |
R827355 (Final) R827355C010 (Final) |
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Dills RL, Zhu X, Kalman DA. Measurement of urinary methoxyphenols and their use for biological monitoring of wood smoke exposure. Environmental Research 2001;85(2):145-158. |
R827355 (2001) R827355 (Final) R827355C010 (Final) |
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Dills RL, Paulsen M, Ahmad J, Kalman DA, Elias FN, Simpson CD. Evaluation of urinary methoxyphenols as biomarkers of woodsmoke exposure. Environmental Science & Technology 2006;40(7):2163-2170. Erratum in: Environmental Science & Technology 2007;41(8):3030. |
R827355 (Final) R827355C010 (Final) |
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Jimenez JR, Claiborn CS, Dhammapala RS, Simpson CD. Methoxyphenols and levoglucosan ratios in PM2.5 from wheat and Kentucky bluegrass stubble burning in eastern Washington and northern Idaho. Environmental Science & Technology 2007;41(22):7824-7829. |
R827355 (Final) R827355C010 (Final) |
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Jimenez J, Wu C-F, Claiborn C, Gould T, Simpson CD, Larson T, Liu L-JS. Agricultural burning smoke in eastern Washington—Part I: Atmospheric characterization. Atmospheric Environment 2006;40(4):639-650. |
R827355 (Final) R827355C003 (2004) R827355C003 (Final) R827355C010 (Final) |
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Larson TV, Gould T, Simpson CD, Liu L-JS, Claiborn C, Lewtas J. Source apportionment of indoor, outdoor, and personal PM2.5 in Seattle, Washington, using positive matrix factorization. Journal of the Air & Waste Management Association 2004;54(9):1175-1187. |
R827355 (2004) R827355 (Final) R827355C003 (Final) R827355C008 (2003) R827355C008 (Final) R827355C010 (Final) R826371C004 (Final) R826788 (2000) R826788 (Final) |
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Simpson CD, Dills RL, Katz BS, Paulsen M, Kalman DA. Determination of levoglucosan in atmospheric fine particulate. Journal of the Air & Waste Management Association 2004;54(6):689-694. |
R827355 (2004) R827355 (Final) R827355C010 (Final) |
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Simpson CD, Paulsen M, Dills RL, Liu L-JS, Kalman DA. Determination of methoxyphenols in ambient atmospheric particulate matter: tracers for wood combustion. Environmental Science & Technology 2005;39(2):631-637. |
R827355 (2004) R827355 (Final) R827355C003 (2004) R827355C003 (Final) R827355C010 (2003) R827355C010 (Final) R829584 (2004) |
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Ward TJ, Hamilton Jr RF, Dixon RW, Paulsen M, Simpson CD. Characterization and evaluation of smoke tracers in PM: results from the 2003 Montana wildfire season. Atmospheric Environment 2006;40(36):7005-7017. |
R827355 (Final) R827355C010 (Final) |
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Wu C-F, Jimenez J, Claiborn C, Gould T, Simpson CD, Larson T, Liu L-JS. Agricultural burning smoke in eastern Washington: Part II. Exposure assessment. Atmospheric Environment 2006;40(28):5379-5392. |
R827355 (Final) R827355C003 (Final) R827355C010 (Final) |
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, ENVIRONMENTAL MANAGEMENT, Air, Geographic Area, Scientific Discipline, Health, RFA, PHYSICAL ASPECTS, Susceptibility/Sensitive Population/Genetic Susceptibility, Toxicology, Risk Assessment, Risk Assessments, Air Pollutants, genetic susceptability, Northwest, Health Risk Assessment, Physical Processes, Epidemiology, Air Pollution Effects, air toxics, Atmospheric Sciences, Biochemistry, particulate matter, Environmental Chemistry, State, aerosols, exposure assessment, California (CA), exposure and effects, environmental hazard exposures, ambient air quality, cardiovascular disease, health effects, inhalation, mortality, epidemelogy, air quality, cardiopulmonary response, hazardous air pollutants, atmospheric aerosols, cardiopulmonary responses, human health risk, particle exposure, toxics, mortality studies, biomarker based exposure inference, acute cardiovascular effects, biostatistics, dose-response, human health effects, particulates, sensitive populations, ambient particle health effects, air pollution, atmospheric chemistry, exposure, biomarker, human susceptibility, ambient aerosol, health risks, human exposure, Human Health Risk Assessment, morbidity
Progress and Final Reports:
2002 Progress Report
2003 Progress Report
Original Abstract
Main Center Abstract and Reports:
R827355 Airborne PM - Northwest Research Center for Particulate Air Pollution and Health
Subprojects under this Center:
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R827355C001 Epidemiologic Study of Particulate Matter and Cardiopulmonary
Mortality
R827355C002 Health Effects
R827355C003 Personal PM Exposure Assessment
R827355C004 Characterization of Fine Particulate Matter
R827355C005 Mechanisms of Toxicity of Particulate Matter Using Transgenic Mouse Strains
R827355C006 Toxicology Project -- Controlled Exposure Facility
R827355C007 Health Effects Research Core
R827355C008 Exposure Core
R827355C009 Statistics and Data Core
R827355C010 Biomarker Core
R827355C011 Oxidation Stress Makers