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Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
EVALUATION OF THE OXIDATIVE POTENTIAL OF URBAN PM AND ITS RELATION TO
in
vitro
INDUCED DNA DAMAGE: A SPATIAL AND TEMPORAL COMPARISON
Raúl Omar QUINTANA-BELMARES
1,2
*, Ernesto ALFARO-MORENO
1
,
Claudia María GARCÍA-CUÉLLAR
1
, Virginia GÓMEZ-VIDALES
3
, Inés VÁZQUEZ-LÓPEZ
1
,
Manuel de Jesús SALMÓN-SALAZAR
3
, Irma ROSAS-PÉREZ
2
and
Álvaro Román OSORNIO-VARGAS
4
1
Laboratorio de Toxicología Ambiental, Subdirección de Investigación Básica, Instituto Nacional de Cancero-
logía. Av. San Fernando 22, Col. Sección XVI, Del. Tlalpan, C.P. 14080, México D.F., México
2
Centro de Ciencias de la Atmósfera, Universidad Nacional Autónoma de México. Circuito de la Investigación
s/n, Ciudad Universitaria, Coyoacán, C.P. 04510, México D.F., México
3
Instituto de Química, Universidad Nacional Autónoma de México. Circuito de Exterior s/n, Ciudad Univer-
sitaria, Coyoacán, C.P. 04510, México D.F., México
4
Department of Paediatrics, University of Alberta, Edmonton, AB Canada. 3-591 Edmonton Clinic Health
Academy, 11405 87
th
Avenue. Edmonton T6G 1C9, Canada
* Autor para correspondencia: qbro@hotmail.com
(Recibido abril 2014; aceptado noviembre 2014)
Key words: PM
10
, PM
2.5
, electron paramagnetic resonance, oxidative potential, DNA damage
ABSTRACT
Some toxic effects of particulate matter (PM) are related to the oxidative potential
(OP) of the particles. The electron paramagnetic resonance (EPR) technique was used
to evaluate the intensity of paramagnetic species (PS) and EPR plus spin trapping,
to evaluate the OP of PM. We evaluated, in parallel, the DNA degradation potential
of PM
10
and PM
2.5
collected from three regions of Mexico City in 1991 and 2003.
Each region had different sources of pollution: industrial, commercial or residential.
Both techniques evaluated Fenton-type reactions in the presence and absence of de-
feroxamine (DFO). PM
10
samples from the industrial region presented similar high
OP, independently of sampling year. PM
10
and PM
2.5
collected in
the commercial
and residential regions in 2003 had similarly low OP. The OP induced by PM
10
from
the industrial region was completely inhibited by DFO, and DFO partially inhibited
the OP induced by PM
10
from other regions. PM
2.5
OP was not inhibited by DFO.
PM from the industrial region was the most potent inductor of DNA degradation,
while PM from residential region was the least potent, correlating with the OP. DFO
inhibited the degradation of DNA induced by PM. The OP of PM collected in the
industrial and residential region correlated with the DNA degradation. The region,
size and year of PM collection are linked to observed OP variations and DNA deg-
radation induced by PM.
Palabras clave: PM
10
, PM
2.5
, resonancia paramagnética electrónica, potencial oxidativo, daño ADN
Rev. Int. Contam. Ambie. 31 (2) 145-154, 2015
R.O. Quintana Belmares
et al.
146
RESUMEN
Algunos de los efectos tóxicos atribuibles a las partículas atmosféricas (PM) están relacio-
nados con su potencial oxidante (OP). La resonancia paramagnética electrónica (EPR) es
una técnica que se utilizó para evaluar la intensidad de las especies paramagnéticas (PS),
y la EPR más un atrapador de espín, para evaluar el OP de las PM. Se evaluó en paralelo
el potencial de degradación de ADN por PM
10
y PM
2.5
muestreadas en tres regiones de
la Ciudad de México en 1991 y 2003. Cada región tenía diferentes fuentes de contamina-
ción: industrial, comercial o residencial. Ambas técnicas fueron evaluadas en reacciones
de tipo Fenton en presencia y ausencia de deferoxamina (DFO). Las PM
10
de la región
industrial presentan alto OP, independiente del año de muestreo. Las PM
10
y PM
2.5
muestreadas en las regiones comercial y residencial durante 2003 tuvieron bajo OP.
El OP inducido por las PM
10
de la región industrial fue completamente inhibido por la
DFO, el OP inducido por las PM
10
procedentes de las otras dos regiones fue inhibido
parcialmente. El OP de las PM
2.5
no fue inhibido por la DFO. Las PM
10
de la región
industrial fueron el inductor más potente de la degradación del ADN, mientras que las
PM
10
de la región residencial fueron las menos potentes, lo que se correlaciona con
el OP. La DFO inhibe la degradación del ADN inducida por las PM. El OP de las PM
de la región industrial y residencial se correlaciona con la degradación del ADN. La
región, el tamaño y el año de muestreo parecen estar vinculados a las variaciones del
OP y a la degradación del ADN inducido por las PM.
INTRODUCTION
It has been widely demonstrated that particulate
matter (PM) is linked to biological effects such as lung
infammation, blood clotting, and various cardiovas
-
cular effects (Alfaro-Moreno
et al.
2007). Some of
these effects (such as endothelial dysfunction) have
been related to the ability of the particles to induce
oxidative stress (Montiel-Dávalos
et al.
2010). Several
studies indicate that different physical and chemical
properties of the particles are related to the intensity
of the biological effects (Veranth
et al.
2006, Rosas-
Pérez
et al.
2007, Mugica
et al.
2009). Differences in
composition seem to be critical, it has also been shown
that PM collected in different cities and even in differ-
ent locations within the same city, show different toxic
potentials (Alfaro-Moreno
et al.
2002, Gerlofs-Nijland
et al.
2009, Osornio-Vargas
et al.
2011).
The toxicological evaluation of PM is a time-
consuming effort. Considering the wide range of PM
composition and size to which humans can be exposed,
there is a need to predict potential toxic effects of PM.
For this purpose, electron paramagnetic resonance
(EPR) has been used to evaluate the oxidative potential
(OP) of PM (Valavanidis
et al.
2005b). Source-related
variations in chemical components and composition
may lead to different oxidative characteristics (Briede
et al.
2005). Therefore, PM generated by different
sources may have a different pattern of oxidative
potential. The relationship between the oxidative
potential’s ability to induce an effect at a biological
level and the oxidation of biomolecules can help to
predict toxicity. EPR can be employed as an alterna-
tive method of predicting the toxic potential of PM in
a quick, sensitive and reliable way.
We have already mentioned that PM represents a
complex mixture and some components, such as met-
als, can cause infammatory processes and increased
reactive oxygen species (ROS), related to Fenton
reactions (Osornio-Vargas
et al.
2011).
It has been shown that different biological pro-
cesses induced by the particles of Mexico City are
associated with PM chemical composition (Alfaro-
Moreno
et al.
2002). One of these effects is DNA
damage and is suggested that DNA degradation oc-
curs as a result of metal content, and the generation
of ROS involving the Fenton reaction (Lloyd 1997,
Lloyd and Phillips 1999).
Naked DNA and the chelating Deferoxamine
(DFO) have been used as a simpli±ed method ²or
evaluating the participation of metals present in PM
and the induction of DNA damage (García-Cuéllar
et al.
2002).
Oxidative stress has been identi±ed as a key
factor in causing biological effects induced by PM.
Although, it is known that cells exposed to PM show
a dramatic increase in oxidative activity (Soukup
et
al.
2000, Becker
et al.
2005), the intrinsic oxidative
potential of PM is not well understood. Furthermore,
it has been shown that PM with a high oxidative
potential is more toxic (Wessels
et al.
2010). In the
present study, we evaluated the signal intensity of
OXIDATIVE POTENTIAL OF PM AND ITS RELATION TO DNA DAMAGE
147
paramagnetic species and the oxidative potential of
urban PM from Mexico City collected in different
regions and years using EPR.
MATERIALS AND METHODS
PM
10
and PM
2.5
sampling
Particulate matter with an aerodynamic diameter
≤10 μm (
PM
10
) was obtained from industrial (IR),
commercial (CR), and residential (RR) regions of
Mexico City using a high-volume sampler (GMW
model 1200 VFC HV PM
10
; Sierra Andersen,
Smyrna, GA, USA) for particles with an aerody-
namic diameter ≤ 10 µm (PM
10
). In 1991, 24 h PM
10
samples were collected using fberglass flters (type
A/E glass 61638; Gelman Sciences, Ann Arbor,
MI, USA; 1.13 m
3
/min), three days per week dur-
ing each week of the year. PM was recovered from
the membranes after dry sonication for 45 min and
subsequently smoothly swept with a brush into an
endotoxin-Free ±ask. All PM samples were pooled
by region and stored dry in endotoxin-free glass
vials, which were kept in a dryer at 4 ºC until use
(Alfaro-Moreno
et al.
2002). In 2003, PM
10
and
particulate matter with an aerodynamic diameter
g ≤ 2.5 μm (
PM
2.5
) were collected using the same
instrument, but we introduced a modifcation in
-
volving the use of cellulose nitrate membranes with
a nominal pore size oF 3 µm. Membranes were cut
from rolls (11302-131, Sartorius, Goettingen, Ger-
many) and modifed to preserve the air±ow rate and
particle size sampling performance, as previously
described (Alfaro-Moreno
et al.
2009).
Determination of the relative intensity of para-
magnetic species in the PM
To evaluate the linearity of the method, we evalu-
ated the relative intensity of the paramagnetic species
using PM
10
(1, 2, 3, 4 and 5 mg) samples from 2003
with an EPR spectrometer (Jeol JES TE-300, Tokyo,
Japan) under the following experimental conditions:
center feld 335 mT; microwave Frequency 9.4 GHz;
microwave power 1 mW; sweep width +/– 4 mT;
sweep time 2 min; modulation width 0.16 mT; am-
plitude 63; time constant 0.3 s and accumulation 1
(
Fig. 1a
).
The relative intensity was obtained in relation to
a standard curve of Tempol by double integration
of each spectrum (Shinji
et al.
2004, Dos Santos
et
al.
2009), using Esprit 382 series V 1.916 Jeol soft-
ware. The results show the signal intensity and are
expressed in arbitrary units.
Relative intensity in the solid PM
To determine the relative intensity of paramag-
netic species in the solid PM samples, 3 mg of
each sample was weighted and evaluated under the
conditions previously described. Two independent
measurements were taken for each PM sample. The
fnal intensity was calculated based on the standard
curve described above.
Evaluation of the oxidative potential of PM
The production oF the hydroxyl radical (•
OH
)
by PM
10
and PM
2.5
was evaluated in the presence
of H
2
O
2
. For this purpose, a suspension of PM was
prepared (3 mg/mL). A total oF 100 µL oF the suspen
-
sion was mixed with 200 µL oF 5,5-Dimethyl-1-Pyr
-
roline N-oxide (DMPO) (fnal concentration 0.1 M,
Dojindo, Rockville, MD), for use as a spin trap,
and 100 µL oF H
2
O
2
(fnal concentration 0.125 M,
Fluka-Aldrich, St. Louis, MO). The mixture was
incubated for 15 min at 37 ºC with continuous
shaking. The sample was fltered (0.2 µm; Ministar-
RC Syringe, Sartorius, Goettingen Germany) and
transFerred to a quartz ±at cell For the EPR measure
-
ments. The Formation oF hydroxyl radical (•OH)
was detected as a well-characterized 1:2:2:1 pattern
of DMPO-OH adduct (
Fig. 1b
; Rinalducci
et al
.
2004) with a
N
=a
H
= 1.49 mT, g = 2.0056 under the
Following experimental parameters: center feld =
335 mT, microwave frequency = 9.4 GHz, micro-
wave power = 1 mW, sweep width +/– 5 mT, sweep
time 0.5 min, modulation width 0.04 mT, amplitude
100, time constant 0.1 s and accumulation 3. A mix-
ture of DMPO (0.1 M in water), phosphate buffered
saline (PBS) and H
2
O
2
(0.125 M in PBS) was used
as a blank (Knaapen
et al.
2000, Shi
et al.
2003).
Inhibition of the oxidative potential of PM
Under the same experimental conditions de-
scribed above we added DFO to the samples as an
iron-copper chelating agent. A total oF 100 µL oF the
PM suspension was mixed with 100 µL oF D²O (fnal
concentration 2.5 mM in water). The samples were
incubated for 3 h with continuous shaking.
Evaluation of DNA degradation
The degradation of “naked” DNA was evaluated in
DNA isolated from Balb/c 3T3 cells with a commercial
kit (DNA isolation kit for cells and tissues; Boehringer,
Mannheim, Germany) as previously described (Gar-
cía-Cuéllar
et al.
2002). A total of 400 ng of isolated
DNA was exposed to 40, 80 or 160 μg/mL oF PM
10
in
the presence of 1 mM H
2
O
2
(Fenton-type reaction).
To inhibit the DNA degradation related to transition
R.O. Quintana Belmares
et al.
148
metals 1 mM of DFO was added. DNA exposed to
CuSO
4
as well as H
2
O
2
was used as a positive control
for a Fenton-type reaction. DNA was exposed to the
particles for 24 h with constant shaking. The samples
were evaluated by electrophoretic mobility (H5 Hori-
zontal Gel Electrophoresis Apparatus; GIBCO-BRL
Gaithersburg, MD) in 1.5 % agarose gels at 100 V for 3
hours, then stained with ethidium bromide (1.2 mg/mL)
and photographed under UV light using a Kodak
Gel logic 200 imaging system (New Haven, CT). All
gels included DNA size markers and the following
DNA degradation positive and negative controls: 1)
l/
Hind
III (1 µg), 2) 400 ng of DNA alone, 3) 400 ng
of DNA with 1 mM H
2
O
2
, 4) a “Fenton-type reaction”
control using 400 ng of DNA plus 5 mM CuSO
4
with
1 mM H
2
O
2
, and 5) 400 ng of DNA plus 1 mM of
DFO and 1 mM H
2
O
2
.
Statistical analysis
A linear regression analysis was performed for
the spin intensity
vs
. PM mass. The slope and R-
value were calculated for each curve. These curves
were used to calculate the intensity of spin in all
evaluated samples. The data obtained for the different
regions, years and sizes of PM were analyzed using
an ANOVA followed by a Bonferroni test (Stata 7.0,
Windows XP, College Station, TX). Differences were
considered signiFcant when p < 0.05.
RESULTS
Determination of the intensity of paramagnetic
species (spin) in PM
Dry PM samples showed an pseudoisotropic broad
signal (g= 2.2 and ∆H= 68 mT) indicating a high
concentration of paramagnetic species (PS), potentially
explained by the presence of previously identiFed lev
-
els of Fe
+3
, Cu
+2
and VO
2
+
in the PM samples (
Fig. 1a
).
These metals are found in all three regions in differ-
ent proportions, though the IR was found to have the
highest concentrations of metals’ related signal. The
intensity evaluation of paramagnetic species in PM
10
from 2003 showed a linear correlation between PM
mass and intensity under the conditions used for the
measurement. When testing the PM from different
regions the correlations yielded similar slopes and
R-values above 0.99 for all samples (
Fig. 2
).
PS intensity of PM from different years and regions
2
mg
0
0
5.0x10
07
1.0x10
08
1.5x10
08
2.0x10
08
IR
CR
RR
PM
10
2003
Relative Intensity
46
Fig. 2.
Relative intensity correlation to PM mass. A linear cor-
relation between PM mass and intensity was observed
for all PM10 samples collected in 2003. The R-value for
all samples was above 0.99, with a similar slope
100
2000
1500
1000
500
g = 2.2
g = 4.3
–500
–1000
–1500
–2000
0
200
A)
B)
300
Magnetic field (m T)
Magnetic field (m T)
400
500
600
332
334
O
OH
H
N
H
3
C
H
3
C
336
338
Intensity
Fig. 1.
EPR experimental spectra at room temperature for: A) solid PM samples, B) Typical EPR spectrum
of short-lived adduct of hydroxyl radical, the DMPO-OH generated in aqueous solutions of PM
samples in the presence of DMPO and H
2
O
2
OXIDATIVE POTENTIAL OF PM AND ITS RELATION TO DNA DAMAGE
149
PM-related PS intensity varied depending on the
sampling region, the PM size and the year of collection
(p < 0.05). PM
10
collected in the IR during 1991
presented the highest intensity, followed by samples
from the CR (p > 0.05) and the RR (p < 0.05;
Fig. 3a
).
PM
10
collected during 2003 did not yield signiFcant
differences among the sampling sites (
Fig. 3b
). Nev-
ertheless, a reduction in spin intensity was observed
in the IR upon comparison of samples from 1991
and 2003 (p = 0.07). In the IR and the CR, PM
2.5
had
lower PS intensities than PM
10
samples, ~ 20 % of
the PM
10
value (
Fig. 3c
).
Oxidative potential of PM by EPR
PM suspensions in the presence of H
2
O
2
pro-
duced a characteristic 1:2:2:1 DMPO-OH adduct
pattern (hfcc a
N
=a
H
=1.49 mT, g=2.0056;
Fig. 1b
),
indicative of hydroxyl radical (•OH) generation
(Rinalducci
et al.
2004) and indicative of OP. The
OP of PM
10
collected during 1991 was similar for
the IR and the CR and slightly lower for the RR
(
Fig. 4a
; p > 0.05). In contrast, the PM
10
collected
in the IR during 2003 demonstrated a higher oxida-
0.08
AB
C
*
*
+
+
0.08
0.020
0.015
0.010
0.005
0.000
0.08
0.020
0.015
0.010
0.005
0.000
0.06
0.04
0.02
0.00
IR
CR
PM
10
, 1991
PM
10
, 200
3P
M
2.5
, 2003
RR
IR
Region
CR
IR
CR
RR
Relative Intensity
Fig. 3.
Intensity of PM collected in different years and regions. A) PM
10
collected
in 1991 showed a signiFcant difference in the intensity depending on the
region of collection. Particles from the residential region (RR) had a smaller
intensity than those collected in the industrial (IR) and commercial (CR) re-
gions. B) PM
10
collected in 2003 did not show signiFcant differences between
regions of collection. C) PM
2.5
collected in 2003 did not show signiFcant
differences between regions of collection. Differences observed regarding
sampling region, PM size and year of collection were statistically signiFcant.
Values represent means of three independent experiments ± standard error
(SE), ANOVA-Bonferroni test (*, + p < 0.05)
4.0×10
–6
3.0×10
–6
2.0×10
–6
1.0×10
–6
0.0×10
+00
A
*
*
+
+
IR
IR + DFO
CR + DFO
RR + DFO
PM
10
, 1991
RR
CR
Oxidative Potential Intensity
4.0×10
–6
3.0×10
–6
2.0×10
–6
1.0×10
–6
0.0×10
+00
B
*
*
IR
IR + DFO
CR + DFO
RR + DFO
PM
10
, 2003
RR
CR
4.0×10
–6
3.0×10
–6
2.0×10
–6
1.0×10
–6
0.0×10
+00
C
*
IR
IR + DFO
CR + DFO
PM
2.5
, 2003
CR
Regions
Fig. 4.
Oxidative potential of PM. A) PM
10
samples from 1991 have no signiFcant difference in oxidative potential
regardless of the region of sampling. Nevertheless, DFO inhibited the OP of samples collected in the industrial
region (IR), but only partially inhibited the OP from the commercial (CR) and residential (RR) regions. B)
PM
10
samples from 2003 collected in the IR had a stronger OP than those collected in the CR and RR. DFO
almost completely inhibited the OP of the samples from the IR and only partially inhibited the OP from the
CR and RR. C) PM
2.5
samples from 2003 yielded no differences among the regions of collection, and the ad-
dition of DFO did not inhibit the OP of any sample. Values represent means of four independent experiments
± standard error (SE), ANOVA-Bonferroni test, (*, + p < 0.05)
R.O. Quintana Belmares
et al.
150
tive potential than those collected in the CR and RR
(p < 0.05;
Fig. 4b
). The OP of the PM
10
from the
IR was signifcantly higher in the samples collected
in 2003 compared to the samples collected in 1991
(p = 0.012). PM
10
and PM
2.5
from 2003 did not yield
signifcant diFFerences in the OP (p > 0.05;
Fig. 4
).
Inhibition of PM oxidative potential by DFO
The OP induced by PM
10
from the IR in 1991 and
2003 was strongly inhibited by the presence of DFO
(p < 0.05;
Fig. 4a
and
b
). In the case of the PM
10
collected in the CR and RR, no signifcant reductions
in the OP were observed. The OP of PM
2.5
was not
inhibited by DFO (
Fig. 4c
).
DNA degradation
All samples of PM were capable of inducing
DNA degradation under the experimental conditions
(
Fig. 5
). In the case of the PM
10
collected in 1991,
the particles from the industrial and the commercial
regions were stronger inducers of DNA degradation
than those collected in the residential region (
Fig. 5a
).
PM
10
samples from 2003 presented a different pat-
tern; those collected in the CR seemed to induce
weaker responses than those from the IR and RR
(
Fig. 5b
). In the case of PM
2.5
, similar effects were
observed independent of the region (
Fig. 5c
). In all
cases, DNA degradation was completely inhibited
by the presence of DFO.
DISCUSSION
In the present study, we observed that the intensity
of the paramagnetic species and oxidative potential of
urban PM collected in Mexico City varied by year and
site of collection. These parameters correlate with the
ability of the PM to induce DNA degradation
in vitro
.
The broad signal induced by the PM dry samples is
the result of the dipolar coupling of a high concentra-
tion of paramagnetic species, including metals such as
Fe
+3
, Cu
+2
and VO
2
+
(Valavanidis
et al.
2005a). In the
center feld, we observed an isotropic fne signal with
∆H = 0.4 mT and g = 2.0026 that was attributable to
stable organic species that could be semiquinones (data
not shown). Regarding the intensity of the paramag-
netic species, we observed that the PM from the IR of
Mexico City collected in 1991 had a higher content of
this intensity than that observed by PM from the RR.
In contrast, the PM collected during 2003 did not differ
depending on the sampling region. When comparing
PM From both years (1991 vs. 2003) a signifcant
reduction in the paramagnetic species content was
observed For the PM From 2003 oF the IR (p < 0.05),
whereas the PM from the RR showed an increase (p
< 0.05). The paramagnetic species in PM From the CR
remained similar in both years of sampling. It has been
reported that the concentration of different components
in PM from Mexico City varied depending on the year
of collection (Vega
et al.
2004, Bae
et al.
2010). The
rapid growth oF both, population and vehicle ±eet
(about 2 million units) during the period 1991-2003
has increased the production of organic compounds,
PM
10
and PM
2.5
in Mexico City. Besides the growth
of the industry, because about 70 % of non-metallic
mineral industries, primary metals industry, food and
beverage production, are important sources of PM
(SMA-GDF 2008). For example, the content of total
carbon and elemental carbon increased from 1997 to
2002, and these changes were related to increases in
traFfc, as well as industrial and commercial activities
(Vega
et al.
2011).
Previous evaluation of metal content by induc-
tively coupled plasma-atomic emission (ICP-AES)
and carbon content (TOR) in the PM
10
from 1991 and
2003 revealed a higher concentration of metals and
carbon in samples collected in 2003 (Alfaro-Moreno
et al.
2009). Particles from 1991 had a gradient of
metal content in which IR>CR>RR, while samples
from 2003 had the pattern IR>RR>CR. In 1991, we
observed that the carbon content had a pattern of
IR=CR>RR, while in 2003, the pattern observed was
IR>CR>RR (Vega
et al.
2011).
When the oxidative potential of PM was evalu-
ated, we observed that the samples collected in 1991
did not show signifcant diFFerences among regions,
while the PM collected in 2003 demonstrated that
the PM from the IR region had a larger OP than that
of the other two regions. If we compare the samples
from 1991 and 2003, it is notable that the PM
10
from
the IR showed a signifcant increase in oxidative
potential in 2003, while the PM from the other two
regions did not signifcantly vary between the diF
-
ferent years. These differences in oxidative potential
could be related to the two following phenomena:
1) changes in the content of metals, mainly Fe and Cu,
or 2) the oxidation of the sample due to storage time.
This oxidation could increase the signal intensity in
dry samples but still contribute to the OP, as with the
less oxidized species. The second hypothesis could
be supported by the observation that the amount of
paramagnetic species is larger in the 1991 samples
than in the 2003 samples. It makes sense that iron
would be a signifcant contributor to the intensity
of paramagnetic species, considering that Fe
2+
is an
inducer of the Fenton reaction (Granados-Oliveros
OXIDATIVE POTENTIAL OF PM AND ITS RELATION TO DNA DAMAGE
151
Fig. 5.
DNA degradation by PM
10
and PM
2.5
. A) PM
10
collected in 1991 in the industrial (IR) and commercial (CR) regions were
stronger inductors of DNA degradation than those collected in the residential region (RR). B) PM
10
from 2003 collected in
the CR appeared to be weaker than those from the IR and the RR. C) PM
2.5
had similar degradation patterns for particles
collected in different regions. For all cases, DNA degradation was fully inhibited by the presence of DFO
Controls
DFO
{
{
{
IR
PM
10
1991
CR
RR
DF
OD
FO
160
λ/HindIll
Rx Fenton
DNA
DNA + H
2
O
2
DNA + H
2
O
2
+ DFO
160
40
80
160
40
80
160
160
40
80
160
40
80
160
160
40
80
160
40
80
-
++++ ++
-
++++++
-
+ +++ ++
H
2
O
2
(1 mM)
A)
Controls
DFO
{
{
{
IR
PM
10
2003
CR
RR
DF
OD
FO
160
λ/HindIll
Rx Fenton
DNA
DNA + H
2
O
2
DNA + H
2
O
2
+ DFO
160
40
80
160
40
80
160
160
40
80
160
40
80
160
160
40
80
160
40
80
-
++++ ++
-
++++++
-
+ +++ ++
H
2
O
2
(1 mM)
B)
Controls
DFO
{
{
IR
PM
2.5
2003
CR
DFO
160
λ/HindIll
Rx Fenton
DNA
DNA + H
2
O
2
DNA + H
2
O
2
+ DFO
160
40
80
160
40
80
160
160
40
80
160
40
80
-
++++ ++
-
++++++
H
2
O
2
(1 mM)
C)
R.O. Quintana Belmares
et al.
152
et al.
2013), but Fe
3+
could also participate. The
nature of the reactions for the generation of hydroxyl
radicals by ferric and ferrous salts in the presence of
hydrogen peroxide has been studied (Yamazaki and
Piette 1990), and it is accepted that the •OH free
radical could be generated from ferric salt according
to the mechanism proposed by Croft
et al.
(1992).
Fe
+3
+ H
2
O
2
Fe
+2
+ HO
2
+ H
+
(1)
Fe
+3
+ HO
2
/ O
2
•–
Fe
+2
+ O
2
+ H
+
(2)
Fe
+2
+ H
2
O
2
Fe
+3
+ HO
+ •OH
(3)
Fe
2+
is not a paramagnetic species, in contrast
to Fe
3+
(Valavanidis
et al.
2009, Gilch
et al.
2010).
Given this, it is likely that the formation of the hy-
droxyl radicals detected comes from both oxidation
states of Fe. In addition to Fe, Cu
+2
is a good promoter
of Fenton-like reactions (Valenzuela
et al.
2008) and
has shown an even higher rate of oxidizing ability
than Fe (Strlič
et al.
2003).
The roles of Fe
2+
and Fe
3+
were highlighted by
the suppression of the oxidative potential when DFO
was added to the PM-DMPO suspension, considering
that DFO is primarily an iron chelator (Valgimigli
et
al.
2001, Karlsson
et al.
2005, Shi
et al.
2006).
Various authors have shown the relationship be-
tween the oxidative potential of PM and biological
effects by measuring lipid peroxidation (Shi
et al.
2006), DNA damage (García-Cuéllar
et al.
2002,
Sánchez-Pérez
et al.
2009, Wei
et al.
2009) and
cellular death (Chirino
et al.
2010). In the present
study, we observed that the intensity of DNA deg-
radation by the Fenton-type reaction correlated with
the oxidative potential. Interestingly, we observed
that the addition of DFO to the mixture of DNA and
PM abolished the degradation of DNA, while the
oxidative potential was still measured with EPR.
However, metal synergisms exist and the participa-
tion of organics has been described as key issues in
PM induced biological effects (Cooper
et al.
2009).
Due to the design of this study, no full effect of the
organic fraction can be explained, as is in the case
of PM
2.5
. Thus, this result could be explained by dif-
ferences in resolution between the two methods used
(i.e., DNA electrophoresis did not detect minor DNA
alterations; Sánchez-Pérez
et al.
2009).
It has been shown that the OP of PM, as measured
by EPR analysis (•OH radicals), correlates with
DNA damage (Shi
et al.
2006). The present study
supports this previous observation. The reason for
this correlation appears to be the high presence of
metals. Considering that Fe
3+
and Cu
2+
are capable of
inducing a Fenton-type reaction (Veranth
et al.
2006,
Gilch
et al.
2010), it seems logical to conclude that
the oxidative potential of PM, which can be inhibited
by DFO, is mainly related to the predominant transi-
tion metals. Wessels
et al.
(2010) have suggested that
increases in PS could be related to larger OP. As has
been previously discussed, this correlation depends
on the chemical species.
CONCLUSION
In conclusion, the evaluation of paramagnetic
species and oxidative potential by means of EPR
provides information supporting the understanding of
the damage induced by the oxidative potential of PM.
In some cases, the evaluation of the oxidative poten-
tial of PM could help to predict potential toxicity of
these materials. However, PM is a complex mixture
composed of different transition metals, organic and
biological compounds that interact and can produce
additive or synergistic effects. But for this study,
metals are the most important sources of OP in PM
samples. The EPR spin trapping technique allows
us to directly monitor the formation of the •OH free
radical and to conduct DNA degradation analysis.
Together, these approaches can be used as primary
methods for evaluating the toxicity potential of PM.
ACKNOWLEDGMENTS
This study was partially supported by the CONA-
CyT (project 106057). We would like to thank
MSc. Yazmín Segura for her technical support during
the 2003 PM sampling, Dr. Yesennia Sánchez-Pérez,
MSc. Eva Salinas Cortés and MSc. Leticia Martínez
Romero for technical advice in standardizing the
DNA degradation assay.
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