1. 1
Oxidative Stress responses in an Atherosclerosis Mouse Model
Exposed to Different Components of Ambient Ultrafine Particles
Abstract:
Increased exposure to air pollutants, such as particulate matter (PM), is a health
concern for people living close to freeways. There is a question as to what specific components
of PM, such as ultrafine particles (UFP), have a higher tendency of exacerbating cardiovascular
diseases (CVD). To gain further insight about whether exposure to UFP accelerates the
progression of CVD, an animal model susceptible to atherosclerosis was exposed to different
components (semi-volatile vs. non-volatile) of ambient UFP for an 8-week period. Control
animals were exposed to purified particle-free air. The topic of study for this project is that the
semi-volatile fraction of UFP, which is rich in polycyclic aromatic hydrocarbons, will induce
more oxidative stress responses than the non-volatile fraction. Oxidative stress was evaluated
by measuring the levels of lipid and protein oxidation biomarkers, and the levels of the
antioxidant glutathione in the serum. The experiment’s data would appear to support the
hypothesis that the semi-volatile organic components present in ambient particulate matter are
exerting more influence on oxidative stress in the system compared to non-volatile
components, as higher average concentrations of lipid peroxidation are experienced in
exposure that accompany the semi-volatile components of UFP.
Introduction:
Particulate Matter (PM), what are also known as particle pollution, is a complex mixture
of extremely small particles and liquid droplets. PM can be recognized based by traits such as
the size of the particle, the means by which it is formed, the origin of its production, and
chemical composition. PM have been classified to contain polycyclic aromatic hydrocarbons
(PAHs), which are a set of extremely reactive oxygen species, meaning they tend to oxidize
whatever corresponding compound or component in reaction, and steal electrons. The size of
PM is possibly linked to their potential for causing health problems. The small size of these

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particles allows them to be easily inhaled by living organisms. As PM travels into the lungs, it
reacts with the cells involved in the organ and gradually degrades the tissue leading to its loss
of function or death. As described by the EPA, PM can stay afloat in the air we breathe for an
extended time. Ultrafine Particles (UFP), the certain fraction of PM we are analyzing in this
experiment, is produced via the combustion of multiple fossil fuels, such as coal, oil, or
gasoline. It is capable of staying in the atmosphere for several days to weeks and can travel
from a few hundred to a few thousand kilometers from where it was originally produced. Thus,
these particles are more likely to be inhaled by an unsuspecting people, as a show of increases
in heart-related diseases show.
Cardiovascular disease (CVD) is a health condition that harmfully effects the
cardiovascular system and include vascular diseases of the brain and kidney, and peripheral
arterial disease. A leading cause of CVD is atherosclerosis, or hypertension, which is known as a
condition in which an artery wall thickens as a result of the accumulation of fatty materials such
as cholesterol.
PM causes these harmful effects on living tissue via the oxidative stress pathway.
Oxidative stress is defined as an imbalance between the systemic manifestation of reactive
oxygen species within a biological system's ability to readily detoxify the reactive intermediates,
or to repair the resulting damage. Disturbances in the normal redox state of cells can cause
toxic effects through the production of peroxides and free radicals that damage all components
of the cell, including proteins, lipids, and DNA.
The objective of this assay is not to study the health effects of UFP as a whole, but to
measure the effects of UFP by its different components through the oxidative stress pathway.
UFP consists of a non-volatile organic core, we refer to it as the denuded fraction. This core is a
part of the UFP with less of a tendency to vaporize and move into the gaseous state. The other
portion under observance is the semi-volatile, or Particle Free Organic fraction (PFO). This
portion is the part of UFP with a higher tendency to vaporize. These two separate fractions
were tested and analyzed for the levels of oxidative stress they caused in different components
of the cells’ serum.

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Materials and Methods:
The experiment was conducted into two different segments, each measuring a different
component of the UFP. One experiment was conducted in 2009 from May through June,
measuring the biomarkers created by the concentration of non-volatile, or denuded fraction of
UFP. The other experiment was conducted in 2011 from April through June, measuring the
biomarkers created by the concentration of semi-volatile, or PFO fraction of UFP. The mice
were exposed to concentrated particles via whole-body inhalation chambers, and located 100m
downwind of the 110 freeway in southern Los Angeles. The exposure periods ran for five hours
a day, four days a week, for eight weeks. An additional controlled-air group was made for each
experiment, where the mice received particle free air instead of a UFP fraction.
The mice used for the experiment were ApoE-/- mice, which are mice that lack the
properties of the apoliopoprotein E (ApoE) gene. The ApoE gene is a segment of DNA in mice
that helps with the metabolism of lipids in cells, and ApoE-/- mice have been bred through
cross-breeding to create inactivation of the ApoE gene. A mouse model of this kind is
commonly used as a model for human atherogenesis, as well as diverse applications in studies
of lipid transport and metabolism, and makes this model an acceptable method of measuring
biomarkers produced during the experiment.
The biomarkers used for analysis are glutathione, malondialdehyde, and protein
carbonyl. Each of these biomarkers was produced by the oxidative stress pathway.
Glutathione (GSG) is an antioxidant designed to prevent damage to important cellular
components caused by reactive oxygen species such as free radicals and peroxides.
Malondialdehyde (MDA) is produced by the degradation of polyunsaturated lipids by reactive
oxygen species. This compound is a reactive electrophile species that causes toxic stress in
cells. Protein carbonyl is produced by an oxidative modification of proteins, being induced into
structural changes that effect the protein’s function and turnover. Each of these indicators
were used to measure the relative level of oxidative stress present in the serum the research
team collected from the mice post-exposure.

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The glutathione component required the use of blank samples and a standard curve in
the analysis for better readings. This assay was modified from the “Griffith Glutathione” assay.
In the analysis of glutathione, supernatants from the mice serum were collected and analyzed
via an enzymatic assay which was modified from the “Griffith” assay. Standards were made by
adding 10mM GSH to the wells on a micro plate. The micro plate was then incubated for 10
minutes at 30 °C. Afterwards, a reaction mixture was then made combining 10mM of GSSG,
3.36mM NADPH, 7mM of DNTB, 140mM of Na2HPO4, and 76µM of EDTA. The reaction
mixture was added to the set of wells and the serum samples collected in the collections were
color monitored for 6.5 min at 412 nm under the use of a Versamax tunable micro-plate reader.
After the standard was made, the procedures called for a transfer of 50µL of tissue
homogenate to be labeled in the micro centrifuge tubes. Then, 50µL of 10% SSA was added to
the precipitated proteins. The centrifuge tubes were then vortexed. Next, the tray of tubes
was incubated at -20°C for 5 minutes, then incubated at 4°C for 15 minutes immediately
afterwards. Following, they were centrifuged for 10 minutes at 15,800g at 4°C. Later, 10µL of
the solution in each tube were transferred to a well on the plate and read for absorbance at
412nm.
The malondialdehyde reading required the setup of blank samples and the production
of a standard curve in analysis for a colorimetric assay. This assay was modified for our use from
the “Bioxytech MDA-586” assay. We formed a reagent solution (NMPI) by combining a 3:1 ratio
of 18mL of Acetonitrile to 6mL of 100% Methanol. The NMPI was stored at 4°C to keep stable
until used. Tetramethoxypropane was provided as the standard in the place of MDA due to
that malondialdehyde is not stable on its own. The blanks were formed by combining 250
NMPI with 20 µM of TMOP, and collecting 250µL of that sample and adding it to another micro-
centrifuge tube containing 250 µL of NMPI reagent, creating a diluted series of blank samples
for analysis ranging from 20 µM, 10 µM, 5 µM, 2.5 µM, 1.25 µM, 0.625 µM, and 0.3125 µM.
The procedure began with adding 140µL of diluted individual samples each to micro-
centrifuge tubes. Next, 455µL of NMPI reactant was added and vortexed into solution. Then,
305 µL of hydrochloric acid was added to equal 1mL, and was vortexed into solution.

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Afterwards, the micro-centrifuge tubes were incubated at 45°C for 60 minutes. Once the tubes
were taken out of incubation, they were allowed to cool down to room temperature for 5
minutes, then centrifuged at 1,000g for 10 minutes to force the precipitate samples that had
formed to collect at the bottom and allow extraction of the supernatants. When this was
completed, 200µL of each sample were pipetted onto a 96-well plate. The absorbance readings
were measured at 570nm.
An albumin standard was produced by using combining multiple set volumes of BSA (µL)
with a diluent. Nine such blank sets were made in varying proportions, decreasing the final
concentration of fluorphore in each micro centrifuge tube. 2,000 µg/mL of blank A was formed
from 300 µL of BSA stock and 0 µL of diluent. 1,500 µg/mL of blank B was formed from 375 µL
of stock and 125 µL of diluent. 1,000 µg/mL of blank C was formed from 325 µL of stock with
325 µL of diluent. 750 µg/mL of blank D was formed from 175 µL of blank B and 175 µL of
diluent. 500 µg/mL of blank E was formed from 325 µL of blank C and 325 µL of diluent. 250
µg/mL of blank F was formed from 325 µL of blank E with 325 µL of diluent. 125 µg/mL of blank
G was formed from 325 µL of blank F and 325 µL of diluent. 25 µg/mL of blank H was formed
from 100 µL of blank G and 400 µL of diluent. Blank I was formed from 400 µL of diluent,
therefore having no concentration of fluorophore.
The protein carbonyl content in serum samples were measured using a fluorometric
assay modified from “OxiSelect.” 200uM Fluorescein-5-thiosemicarbazide (FTC) was added in a
1:1 ratio with serum samples. Nucleic acids had been removed from the samples by
precipitation with 10% streptomycin sulfate. After overnight incubation, 20% trichloroacetic
acid was added to precipitate the protein. Excess unbound fluorophore was washed away with
3 acetone rinses. The precipitated protein were dissolved in 6 M guanidine hydrochloride and
the fluorescence was read at 485 emission wavelength/ 530 excitation wavelength. Protein
carbonyl concentrations were normalized by total protein concentration in the sample. Protein
concentration was measured using the Pierce Bicinchoninic Acid colorimetric assay (Pierce
Biotechnology, Thermo Scientific, Rockford, IL).

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50µL of each sample are added to a centrifuge tube, and another 50µL of 1X protein
carbonyl fluorophore solution was added to each sample tube, then vortexed. The samples
were then incubated overnight at room temperature and shielded from any light. The next day,
400µL of 1X TCA solution was added to each tube and vortexed thoroughly, then incubated on
ice for 10 minutes. The tubes were then centrifuged at 10,000g for 10 minutes. Afterwards the
supernatant was discarded. 1mL of acetone was added to each centrifuge tube and vortexed
thoroughly in order to break up the pellet. Once again, the tubes are centrifuged at 10,000g for
10 minutes, and then they are rinsed with acetone twice more. Afterwards, the supernatant is
discarded and the pellet formed at the bottom of the tube was allowed to dry for 1 hour. Once
the acetone had completely evaporated, 50µL of protein solubilization solution was added to
each tube, and vortexed for roughly 10 minutes while in incubation at room temperature.
Next, 450µL of the diluted assay diluent was added to each tube and vortexed. The tubes were
centrifuged once more at 10,000 x g for 10 minutes to remove the excess debris. Then 100µL
of each sample were transferred to a 96-well black fluorescence microliter plate, and finally the
readings were analyzed at an absorbance of 485nm/530 excitation wavelength.
Results:
Total antioxidant GSH levels. The blue bar graph shows data for the 2009 exposures and
the red bar graph show the data for the 2011 exposures. The bars represent mean ± standard
error. These graphical representations indicate the total amount of glutathione present in the
serum collected from the apoE -/- mice used in the experiment. The relative concentrations of
glutathione in each group (air, denuded, and PFO) reveals the rate in which GSH is being
reproduced via the enzymatic recycling method in solution. This production is directly
proportional to the 5-thio-2-nitrobenzoic acid (TNB) present in the serum, as GSH is recycled to
produce more of TNB.

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As shown, the GSH µM concentrations of the UFP groups are relatively close within their
ranges of standard deviation when compared with one another. In reflection of the controlled
air group, there appear to be differing slopes of concentration when compared to either the
denuded fraction or PFO fraction. However, because of their close concentrations and
overlapping standard deviations of error, we could attribute these values under non-sequential
error due to another factor not relative to the experiment. The GSH levels seen on these
graphs do not show any positive trend in which oxidative stress has strong influence on the
usage, or presence of glutathione in the serum extracted from the mice.
Serum Lipid Peroxidation is shown by the data in the blue bar graph for the 2009
exposures and the red bar graph for the 2011 exposures. MDA levels were normalized by the
total protein content of the sample. The bars represent mean ± standard error. These
graphical representations indicate the total concentration per milligram of MDA present in the
serum collected from the apoE -/- mice used in the experiment. The relative amounts of MDA
in each group (air, denuded, and PFO) reveals the proportion at which MDA was produced by
lipid peroxidation in serum via oxidative stress.

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According to the graphs, the concentrations of MDA vary significantly within the PFO
group, and the total amount in the denuded group compared with the others. This noticeable
difference is indicated in the amount of MDA present in the semi-volatile fraction and the total
group of denuded, as an asterisk placed on the graph represents a significant statistical
difference from the control group (p<0.05).
In the 2009 group, the total MDA capacity is measured and determined to be
significantly higher than the control and denuded bar graphs present in the same year
exposure. In the 2011 group, that significant statistical difference is now seen in the PFO
exposure. As a result, this data suggests that lipid peroxidation plays a larger role over the
production of MDA in the serum of mice, and is a strong indicator of oxidative stress.
Protein Oxidation levels in serum are represented by the data in the blue bar graph for
the 2009 exposures and the red bar graph for the 2011 exposures. Protein carbonyl levels were
normalized by the total protein content of the sample. The bars represent mean ± standard
error. These graphical representations indicate the total concentration per milligram of protein
carbonyl present in the serum collected from the apoE -/- mice used in the experiment. The
relative amounts of protein carbonyl in each group (air, denuded, and PFO) reveals the
proportion at which protein carbonyl was produced by protein oxidation in serum via oxidative
stress.

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As shown, the protein carbonyl concentrations of the UFP groups are relatively close
within their ranges of standard deviation, as like the data representing GSH. In reflection of the
controlled air group, there appear to be differing slopes of concentration when compared to
either the denuded fraction or PFO fraction. However, because of their close concentrations
and overlapping standard deviations of error, we could attribute these values under non-
sequential error due to another factor not relative to the experiment. The data levels seen on
these graphs do not show any positive trend in which oxidative stress has strong influence on
the presence of protein carbonyl in the serum extracted from the mice.
Discussion/Conclusion:
Of the three different biomarkers analyzed in experimentation, the most recognized
form was the production of MDA via lipid peroxidation. Increased levels of lipid peroxidation in
the serum of animals exposed to total UFP and PFO suggest that lipid modification may be
occurring due to the presence of SVOCs in particles, while mice exposed to denuded particles
did not show increased levels of lipid peroxidation. An observation that provides support for
this is seen as the results for lipid peroxidation matched the trend observed with plaque
accumulation (data not shown). This suggests that lipid modification by SVOCs exposure might
be a mechanism involved in the acceleration of plaque accumulation in atherosclerotic mice.
Overall, the data does not prove the hypothesis, but it does seem to support the study, that the

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semi-volatile organic components present in ambient particulate matter are in fact producing
an effect of oxidative stress in the system compared to denuded portion of UFP, as higher
average concentrations of lipid peroxidation are experienced in exposure that accompany the
semi-volatile counterpart of UFP.
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composition, and oxidative stress. Particle and Fibre Toxicology. 2009. 6:24
2. Simkhovich B.Z., Kleinman M.T., Kloner R.A. Air Pollution and Cardiovascular Injury.
Journal of the American College of Cardiology 2008; 52:719-726.
3. Sun Qinghua, et al. Long-term Air Pollution Exposure and Acceleration of Atherosclerosis
and Vascular Inflammation in an Animal Model. JAMA. 294.23: 3003-3010.
4. Pakbin P., Ning Z., Eiguren-Fernandez A., Sioutas C. Modification of the Versatile Aerosol
Concentration Enrichment System (VACES) for conducting inhalation exposures to semi-
volatile vapor phase pollutants. J Aero Sci. 2011; 42.9: 555-556.
5. Griffith OW. (1980) Determination of glutathione and glutathione disulfide using
glutathione reductase and 2-vinylpyridine. Anal Biochem 106:207-212.
6. Gerard—Monnier D et al. (1998) Reactions of 1-Methyl-2-phenylindole with
malondialdehyde and 4-hydroxyalkenals. Analytic applications to a colorimetric assay of
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in blood plasma. Anal Biochem 400, 289-294
Acknowledgements:
Project Supported By: Louis Stokes Alliance for Minority Participation, and California Air
Resources Board ARB-07-307.
Thank you to all who contributed to the Project:
Dr. Loyda Mendez, Andrew Keebaugh, and Dr. Michael T. Kleinman