BIOANALYTICAL APPROACHES TO POPS DETECTION - POSTERS

Ah RECEPTOR MEDIATED EFFECTS OF PARTICULATE ORGANICEXTRACTS FROM THE PASO DEL NORTE AIRSHED ALONG THE U.S.MEXICO BORDER
Daniel E. Arrieta'. Cynthia Ontiveros', Michael S. Denison^ & Barbara Shayne Washbum^
1 Department of Biological Sciences and Border Biomedical Research Center, University ofTexas,
El Paso, TX 79968^Department of Environmental Toxicology, Meyer Hall, University of California, Davis, CA
95616
Introduction
Particulate matter (PM) is comprised of organic and elemental carbon aggregates containing
various metals, acid salts, and organic pollutants'. Organic compounds adsorbed to PM cancontain carcinogens and mutagens^. Recent epidemiological studies have demonsfrated causal
associations between exposure to particulate matter and an increased risk of lung cancer andadverse affects on fetal development. Studies conducted in many cities around the worldconsistently demonsfrate mutagenicity of PM co-pollutants'*''. The Paso del Norte air basin
encompass three cities along the U.S.-Mexico border; El Paso, TX, Sunland Park, NM, andCiudad Juarez, Chihuahua, Mexico. In the Paso del Norte air basin, PMio levels exceed federal
guidelines, therefore increasing theriskof human exposure to harmful environmental compounds.
Anthropogenic and man-made natural sources of contaminantsfromboth sides ofthe borderconfribute to air pollution within this region. Sources of point and non-point pollutants havebeen identified as vehicular traffic (about 20%), open buming of scrap fuel during winter (40%),
brick kilns, unpaved roads in Juarez (50%), and two refineries (J. Sema, Physical Science
Laboratory, NMSU, pers comm.). In addition, winter inversion layersfrapparticulate matter inthe valley and increase PMio levels.
The specific aim ofthis study is to identify biomarkers which would serve as an indicatorof air toxics in the Paso del Norte airshed. This study will provide baseline data on biologicalalterations associated with exposure to various types of organic contaminants and aid in theidentification of important envfronmental contaminants.
Materials and Methods .
PMIO filters were collected during the winter of 1998-1999fromhigh volume afr
samplers from two sites in El Paso and one site in Juarez. The sites selected in El Paso wereNortheast Clinic, reference site; and Tilhnan Healtii Center, a site with historically high levels ofPMio in downtown. The site selected in Juarez was Advance Transformer located next to atransformer production facility and within 200 yards of a brickmaking district. Fuels used in thebrick kibs includes sawdust, wood pallets,tires,and other scrap fiiel. One-half of the 8" x 10"
filters were extracted individually using a Soxhlet apparatus with 100 ml of dichloromethane(DCM) for 24 h, followed by concentration with a Kudema-Danish apparatus (EPA method3450C). Samples were stored in DCM in amber vials with PTFE-lined caps at 4°C*.
Afr equivalents (AE)are defmed as the amount of respirable air an average adult breathes
in a specified time period. An average individual exchanges approximately 22-24 mVday'.
Measurement of CYPI A1 activity was measured in H41IE cells cultured according to Sanderson
et. al. and exposed various amounts of organic exfracts resuspended in DMSO (fmal concenfration
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BIOANALYTICAL APPROACHES TO POPS DETECTION - POSTERS0.5%). Following exposures, cells were rinsed with Hanks balanced salt solution, lysed withmammalian protein extraction reagent (Pierce, Rockford, IL), homogenized and cenfrifiiged at 160
X g for 10 minutes at 4°C. Supematants were collected and stored in buffer with 20%glycerol at 20°C. Ethoxyresomfin O-deethylase (EROD) activity was measured fluormetrically.
Because compounds present in organic extt'acts might inhibit EROD activity*, we also
used the chemically activated luceriferase expression (CALUX) bioassay to evaluate PM samples.
Methods for culturing recombinant mouse cells and measuring luciferase activity followed Ziccardi
et. al.'.
To assess toxicity of exfracts over a range of air equivalents, XTT assays wereperformed. Both HII4E and recombmant mouse hepatoma cells were tested with 1 and 10 minutesof air equivalents. Cells were measured specfrophotomefrically at 450 nm to determine cell
viability.
Results and Discussion
Dose response relationships were determmed for PM exfracts and EROD and luciferase
induction. For P4501A1 activity in HII4E cells, we observed highest activity at 12 minutes of afr
equivalents, after which inhibition was noted with exfracts from sites with higher particulates. In
contrast, for the AhR-luc reporter system, the response remained linear between 30 seconds and
90 minutes of air equivalents.
Cytotoxicity assays were performed to ensure that the air equivalent dose used in theexperiments does not cause overt toxicity. We found no cytotoxicity in HII4E cells exposed to 10
minutes afr equivalents. Cytotoxicity measurements will be made in recombinant mouse cells
exposed to 1 minute AE.
The highest EROD activity was observed in cells exposed to extracts from the Advancesite. The Advance PM sampling site is situated in Ciudad Juarez within a maquiladora disfrict
immediately adjacent to a brickmaking area. Brickmakers are known to use a variety of scrap fiiels
to fu'e bricks, including tfres, sawdust from indusfrial facilities, woods, and other scrap material.
An initial analysis of chemicals contained in the DCM extract show that the PCB concentration atthe Advance site is approximately 1.1 ng/m' (W. Jarman, U. of Utah, pers. comm.). We
observed similar pattems between EROD activity and amount of particulates collected from any
one date. This suggests that the composition of PM remains fairly constant, regardless oftheamount of particulates collected.
EROD activity associated with the Northeast and Tillman sites was reduced compared toAdvance, suggestmg either lesser amounts of AhR ligands or inhibition by those chemicals. Datafrom the AhR luc assay followed the same pattem as EROD data for all dates, suggesting a lesser
amount of ligands present. However, at the Advance site, luc activity was significantly inducedon Jan 30 whereas EROD activity was moderate (8 pmol/min/mg protein) relative to other dates.
This suggests that significant amounts of Ah receptor ligands acted to inhibit EROD activity.
We examined the relationship between total particulates (g) and the activity ofthe twobiomarkers to determine if they varied in the same fashion. On most dates, when particulate loads
were high, both EROD and LUC activity increased. However, in some instances this was not thecase. For example, on January 30 at the Advance site, PM levels were modest, 0.15 g per filter,
EROD activity was modest (8 pmol/min/mg prot) whereas LUC activity was over 3500 RLU/mg
protein. This would suggest that there are increased amount of AhR ligands probably associatedwith scrap fiiel used in the brick kihis that are inhibiting EROD activity. Further, fuelcomposition probably varies on different dates which accounts for the discrepancy between PM
weight and LUC activity.
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Acknowledgements
This research project is supported by NIGMS Grant# 5612-RR08124 to the Border Biomedical
Research Center at the University ofTexas at El Paso and NIEHS Grant# R15 ES09938-0I7 toB.S.W. Additional support was provided by NIEHS Superfimd Basic Research Grant# ES 04699
to MSD and NIEHS Center Grant# ES 05707. We would like to thank J. Reynoso and H. Del
Rio, El Paso City- County Healtii and Environmental Disttict, and G. Tarin Tortes and R.
Mercado, Departamento de Ecologia, Cd. Juarez for provided us with the PM filters. We
appreciate technical assistancefromSharon Heatii-Paglliaso. We also appreciate the help ofthefollowing undergraduates: Victor Lopez and Aurora Vfrgen.
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Gravimettic data (g) perfilterfor January, 1999from3 sites in the Paso del Norte airshedDate
1-6-991-12-99
1-18-99
1-24-99
1-30-99

Northeast
.07
.06
.031
.043
.04

Advance
.276
.111
.152
.203
.163

Tillman
.101
.057
.047
.087
.043

Luciferase activity(mean +/-s.e.)s Advance• Northeast
D Tillman

1-6

1-12 1-18 1-24 1-30
date

EROD activity(mean +/-s.e.)

w15

I• | S« 10imAd/ance

J oo a eE^Q.

0m Northeast

111^7^1lyiIft1-6

1-12 1-18 1-24
date

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1-30215

D Tillman