Indian Pediatr 2010;47: 233-239
Oxidative Stress and Anti-oxidative Defense in Schoolchildren
Residing in a Petrochemical Industry Environment
A Vujovic, J Kotur-Stevuljevic, D Kornic*, S Spasic, V
N Bogavac-Stanojevic, A Stefanovic, M Deanovic*, S Babka†,
B Aleksic† and
From the Institute of Medical Biochemistry, Faculty of
Pharmacy, Belgrade, *Health Center Pancevo and †Health
Center Kovacica, Pancevo, Serbia.
Correspondence to: Ana Vujovic, Institute for Medical
Biochemistry, Faculty of Pharmacy Vojvode Stepe 450,
POB 146, 11000 Belgrade, Serbia.
Received: August 22, 2008;
Initial review: October 3, 2008;
Accepted: February 27, 2009.
Published online 2009 May 20.
Objective: To evaluate the possible relationship
between industrial air pollution and oxidative stress in schoolchildren
by comparing parameters from children residing in two nearby localities
with contrasting environmental conditions.
Participants: 42 schoolchildren (12-15 years)
from Pancevo (site of Serbia’s largest petrochemical installation)
formed the exposed group. 82 schoolchildren from Kovacica village,
located 30 km north of Pancevo, formed the non-exposed group.
Methods: Oxidative stress status, anti-oxidative
defense parameters, paraoxonase-1 status, lipid status, glucose
concentration and leukocyte counts were compared in two groups.
Results: The children from Pancevo showed higher
level of oxidative stress demonstrated by an elevated malondialdehyde
concentration (P <0.001) and decreased superoxide dismutase
activity (P<0.01) in comparison to the non-exposed group.
Conclusions: The results suggested a relationship
between the presence of air pollutants and increased oxidative stress in
schoolchildren residing in an industrial environment.
Key words: Air pollution, Anti-oxidative defense,
Cardiovascular disease, Environment, Oxidative stress.
any epidemiological studies have
demonstrated air pollution as a risk factor for respiratory illnesses,
malignancies and cardiovascular diseases (CVD)(1-4). Environmental air
contains a range of pollutants, many of which are free radicals or have
the ability to drive free radical reactions. Exposure to these pollutants
gives rise to oxidative stress (OS), which appears to initiate responses
particularly dangerous to children(5-7). An early increase of OS may be
responsible for predisposition to premature atherosclerosis leading to
development of CVD in later life(8-10). There is a paucity of data
evaluating the effect of air pollution on OS and anti-oxidative defense (AOD)
parameters in healthy children. It is known that the Serbian males have
increased risk for early development of CVD and its consequences(11). We
planned this study with the objective of evaluating the contribution of
environmental air pollutants on OS status parameters and on protective
antioxidative high-density lipo-protein (HDL) function in Serbian
Study population and sample size
The study population consisted of 124 healthy
schoolchildren (aged between 12 and 15 years) scheduled for a regular
health check in April and May 2007. Forty two (34%) children were from
Pancevo where Serbia’s largest petrochemical facilities and oil refineries
are situated. These individuals were labeled as ‘exposed group’ (EG). The
living conditions in Pancevo are characterized by a high concentration of
chemical industries, and the close proximity of industrial zones to
residential areas(12). Eighty two children from Kovacica village, located
30 km north of Pancevo, formed the ‘non-exposed group’ (NEG). The main
occupation of parents in the municipality of Kovacica is agriculture.
Dietary habits are similar for both locations.
Sample size was determined according to Hopkins(13),
using the formula N=32/ES2
where ES is the smallest effect size worth detecting (smallest difference
worth noticing divided by standard deviation, expressed in the same
units). The ES for our study was 0.55. With Type I error of 0.05 and Type
II error of 0.2, the sample size was calculated as 119 with unequal number
of subjects in two groups (39+80). Informed consent was obtained from
children and their parents prior to enrolment in the study. The
institutional review committee of the Faculty of Pharmacy, University of
Belgrade and committees of both Health Centers’ approved the study
Air pollution monitoring in Pancevo
Air pollution in Pancevo was determined according to
standard operating procedures(14) from the air pollution monitor network.
The mean concentrations of benzene, total suspended particles, polycyclic
aromatic compounds and benzopyrenes were more than the maximum allowable
concentrations (MAC) on most days in three years preceding the survey,
whereas the levels of sulphur dioxide, nitrogen dioxide, ammonia, toluene
and total precipitated matter were below the MAC on all days during this
period. Air pollution data from Kovacica were not available as it is not
monitored because of its distance from industries.
Blood sampling and analysis
Venous blood was drawn from the antecubital vein after
overnight fasting and samples were stored at – 80° C in aliquots, until
analysis. For malondial-dehyde (MDA) determination, butylated hydro-xytoluene
(BHT, 0.05% w/v) was immediately added to the plasma (0.2 mL) before
storage, and for the measurement of superoxide anion (O2-),
plasma from heparinized blood samples was used immediately. Leukocyte
count was determined by a combination of flow impedance and light
absor-bance using a Pentra 60C+ analyser (Horiba ABX, Montpelier, France).
Glucose, total cholesterol (TC) and triglycerides (TG) were assayed by
routine enzymatic methods using an ILab 300+ analyser (Instrumentation
Laboratory, Milan, Italy) and Randox Laboratories (Armdore, UK) reagents.
High-density lipoprotein cholesterol (HDL-C) was measured using the same
method (as above) after precipitation of the plasma with phosphotungstic
acid in the presence of magnesium ions. Apolipoprotein A-I (apoA-I) and
apolipoprotein B (apoB) were measured by immunoturbidimetry using the ILab
600 analyser and Dialab (Vienna, Austria) reagents. The concentration of
low-density lipoprotein choles-terol (LDL-C) was calculated using the
Friedewald formula(15). The plasma concentration of lipo-protein(a) [Lp(a)]
was measured using immuno-turbidimetry (BIOKIT, Barcelona, Spain). The
lipid tetrad index (LTI) was calculated using the formula [TC × TG × Lp(a)]/HDL-C(16)
and the lipid pentad index (LPI) was calculated using the formula [TC × TG
× Lp(a) × apoB]/apoA(17).
Parameters of oxidative stress
To determine the OS index, we used the thiobarbituric
acid-reacting substances (TBARs) assay that measures the quantity of the
malondialdehyde (MDA)-TBA 1:2 adduct described previously by Girotti,
et al.(18). The rate of nitroblue tetrazolium reduction was used to
measure the level of O2-,
as previously described(19). Plasma super-oxide dismutase (SOD) activity
was measured according to a previously published method(20). The method
for plasma lipid hydroperoxide (LOOH) determination is based on the
oxidation of Fe2+ to Fe3+ under acidic
conditions(21). The plasma advanced oxidation protein product (AOPP)
concentration was determined using standard method(22).
Determination of Paraoxonasi-1 (PON1) status involved
the measurement of PON1 activity towards two substrates [paraoxon (POase
activity) and diazoxon (DZOase activity)] and the subsequent assessment of
phenotype. Rates of POase and DZOase activity were measured
spectrophotometrically using a UV/VIS Ultrospec III spectrophotometer
(Pharmacia LKB, Cambridge, UK) in serum according to the method described
by Richter and Furlong(23,24). The PON1192 pheno-type (QQ, QR
or RR) was predicted after examination of the two-dimensional plot of
diazoxon vs. paraoxon hydrolysis rates and also by calculating the DZOase/POase
The differences between the groups with continuous
variables were statistically tested using the Student’s t test for
normally distributed variables. As the distributions of TG, Lp (a), LTI,
LPI, HDL-C, POase, O2-,
MDA, AOPP and the O2- /SOD ratio were skewed, logarithmic
transformation of the values was performed before statistical comparisons.
The distribution of LOOH concentrations was not normally distributed even
after logarithmic trans-formation so they were compared using the
Mann-Whitney test. Because obesity can influence the OS status(25), the
BMI (body mass index) was used for analysis of covariance in order to
determine the adjusted means. Categorical variables and phenotype
distributions between the study groups were compared using the Chi-square
test. Two-tailed P values less than 0.05 were considered
statistically significant. All analyses were conducted using MedCalc® (Mariakerke,
Belgium) version 9.3.90.
Basic demographic and biochemical characteristics of
the two examined groups are shown in Table I. 22
subjects in exposed group (EG) and 37 in non-exposed group (NEG) were
males. The mean age and body mass index of children in NEG were
significantly greater than those in EG. There were no significant
differences in the basic biochemical parameters between the two groups.
Basic Demographic Characteristics and Biochemical Parameters of the Study Groups
||Exposed (n=42), mean (SD)
||Non-exposed (n=82), mean (SD)
Lp a (mg/L)*
||4.6 x 103 [(3.0-7.0)x103]
||4.0 x103 [(3.1-5.2) x103]
||1.9x 102 [(1.3-2.8) x102]
||1.6 x 102 [(1.2- 2.0) x 102]
Glucose ( mmol/L)
BMI, body mass index; TC, total cholesterol; TG, triglyceride; LDL-C, low-density lipoprotein cholesterol;
HDL-C, high-density lipoprotein cholesterol; apoA, apolipoprotein A-I; apoB, apolipoproteinB; Lp(a), lipoprotein(a);
LPI, lipid pentad index; LTI, lipid tetrad index;
* Mean values derived from log normal distribution given as geometric mean values (95% CI).
The EG exhibited a higher level of OS (demonstrated by
an elevated MDA concentration) when compared to the NEG (Table
II). SOD activity was significantly lower in the EG compared with the
NEG (P=0.043). DZOase and POase activities showed no significant
differences between the two groups, although in the EG both enzymatic
activities were lower. The adjusted MDA concentration in the EG was
significantly higher than that in the NEG (P=0.002).
Oxidative Stress Status and Anti-Oxidant Defense Parameters in the Study Groups
||Exposed (n=42), mean (SD)
||Non-exposed (n=82), mean (SD)
||381 (320 - 455)
O2-, superoxide anion; MDA, malondialdehyde; AOPP, plasma advanced oxidation protein product;
LOOH, lipid hydroperoxide; SOD, superoxide dismutase; O2- /SOD, OS index; POase, PON1 activity towards paraoxon;
DZOase, PON1 activity towards diazoxon.
* Mean values derived from lognormal distribution given as geometric mean values (95% CI);
†Values presented as median (interquartile range),P value for Mann-Whitney test.
There was no statistically significant difference in
the PON1192 phenotypes
between the two groups (QQEG vs. QQNEG 0.49
vs. 0.47; QREG vs. QRNEG 0.42 vs.
0.43 and RREG vs RRNEG 0.09 vs. 0.10,
P=0.832). In order to further analyze changes in PON activities, we
categorized children from both groups according to their PON1192
phenotype and compared corresponding POase and DZOase activities. Distinct
PON1 phenotype activity was always lower in the EG compared to the NEG.
The highest statistical significance was found in the RR phenotype
subgroup (Table III).
PON1 Activity Toward Paraoxon and Diazoxon According to PON1 Activity Phenotypes
|| POase activity, Geometric mean (95%
||DZOase activity, (mean±SD)
POase, PON1 activity towards paraoxon; DZOase, PON1 activity towards diazoxon; QQ, QR,RR,
phenotype subgroups for PON1192 isoenzyme.
The increase in CVD in Serbian adults(11) and the fact
that risk factors are established at a young age indicate the necessity to
investigate possible contributors in young populations. We
evaluated the potential influence of outdoor air pollution on OS in
schoolchildren in two nearby localities having distinct living
Total suspended particles (TSP) of smaller size have
been shown to penetrate the alveolar epithelium causing local inflammation
and OS. The systemic inflammatory response to particulate air pollution
and its relationship to adverse coronary events in patients with coronary
artery disease and in the healthy population are well established(4,6,25).
Polycyclic aromatic hydrocarbons (PAH) can induce OS indirectly through
its biotransformation by liver enzymes to generate redox active quinones
which act as catalysts for free radical production(5,6). In vitro
reactive oxygen species (ROS) formation has been shown to be highly
correlated with PAH content in air(6). The oxidation of membrane lipids,
one of the primary events during oxidative cellular damage, can be
assessed by measuring plasma MDA concentrations, a late-stage OS biomarker
of injured cells and subcellular structures(26,27).
Our finding that increased MDA concentrations in children belonging to the
EG was consistent with a study by Zalata, et al.(28).
In our study, the LOOH level, a marker of early damage
to cellular membranes, lipoproteins and other lipid containing
molecules(21,29) did not significantly differ between the two groups.
However, an elevated MDA concentration(10) and a tendency for AOPP to
increase(30) in the EG indicated long term (chronic) exposure to air
pollution in Pancevo.
Protective HDL function is primarily via the enzyme
paraoxonase 1 (PON1) which hydrolyzes lipid peroxides in human
atherosclerotic lesions(31-33). PON1 has a common coding region
polymorphism, a glutamine (Q) to arginine (R) substitution at position
192, which is associated with a number of pathophysiological conditions
such as CVD and some others(31). It is well-documented that PON1192RR
isoform is less capable for antioxidative protection than PON1192QR
or PON1192QQ iso-forms(32). The activities of PON1 and SOD were
analyzed as indicators of the AOD status. Significantly lower plasma SOD
activity, which indicated a reduced ability of SOD to remove ROS, was
noted in EG. We detected reduced PON1 activity in children exposed to air
pollution, although this difference did not reach statistical
significance. Lower POase and DZOase activities in children from the EG
was particularly evident and reached statistical significance in PON1192RR
risk phenotype carriers.
A limitation of our study was the absence of air
pollution related data in the Kovaica village, which we assumed as
non-exposed group. It would have been interesting to see whether the air
pollution levels are actually less at a distance of 30 Km from industries
but the absence of monitoring in this village precluded this analysis.
Children from NEG had significantly greater BMI and they were
significantly older than children from EG but they were not in the state
of OS. Greater BMI and older age could lead to higher OS parameters and
lower AOD parameters values(34). On the contrary, children from EG, even
younger and thinner, were in OS state.
In conclusion, there is an enhanced oxidative stress
and a fall in anti-oxidant defense in children exposed to industrial
pollution. Increase in serum MDA concentration and decrease in plasma SOD
activity in the exposed children could be an indirect response of cells to
increased oxidative challenge attributable to air pollution. These
findings support the view that air pollution increases oxidative stress
and suggest a metabolic self-defense adaptation mechanism of antioxidant
systems following exposure to air contaminants.
Verica Milanovic and Marina Baranin for their support
with the laboratory analyses, and David R Jones for help in editing the
Contributors: AV performed
experimental work, statistically analyzed data and wrote the manuscript.
JKS, DK and AS performed experimental work. SS, JKS and NBS participated
in study design and statistical analysis. DK, MD, SB and BA helped in data
collection. SS, JKS, NBS and ZJI critically revised the manuscript.
Funding: Ministry of Science and
Environmental Protection, Republic of Serbia (Project number 145036B).
This study was also supported by cost B35 Action.
Competing interests: None stated.
What is Already Known
• Oxidative stress and weakened antioxidative
defense are factors in cardiovascular disease pathogenesis and
What this Study Adds
• Healthy children living near petrochemical
industries in Serbia have increased oxidative stress and decreased
anti-oxidant defense mechanism.
1. Ballester F, Rodríguez P, Iñíguez C, Saez M, Daponte
A, Galán I, et al. Air pollution and cardiovascular admissions
association in Spain: results within the EMECAS project. J Epidemiol
Commun Hlth 2006; 60: 328-336.
2. Health aspects of air pollution. Results from the
WHO project "Systematic Review of Health Aspects of Air Pollution in
Europe": Report of a WHO Working Group. Copenhagen, WHO Regional Office
for Europe, 2004. Available from: URL: http://www.euro.who.int/document/E83080.
pdf. Accessed on December 3, 2007.
3. Pope CA III, Burnett RT, Thun MJ, Calle EE, Krewski
D, Ito K, et al. Lung cancer, cardio-pulmonary mortality and
long-term exposure to fine particulate air pollution. JAMA 2002; 287:
4. Brook RD, Frankin B, Cascio W, Hong Y, Howard G,
Lipsett M, et al. Air pollution and cardiovascular disease. A
statement for healthcare professionals from the Expert Panel on Population
and Prevention Science of the American Heart Association. Circulation
2004; 109: 2655–2671.
5. Kelly FJ. Oxidative stress: its role in air
pollution and adverse health effects. Occup Environ Med 2003; 60: 612-616.
6. Li N, Sioutas C, Cho A, Schmitz D, Misra C, Sempf J,
et al. Ultrafine particulate pollutants induce oxidative stress and
mitochondrial damage. Environ Health Persp 2003; 111: 455-460.
7. Schwartz J. Air pollution and children’s health.
Pediatrics 2004; 113: 1037-1043.
8. Raitakari OT, Juonala M, Kahonen M, Taittonen L,
Laitinen T, Maki-Torrko N, et al. Cardiovascular risk factors in
childhood and carotid artery intima-media thickness in adulthood: the
Cardiovascular Risk in Young Finns Study. JAMA 2003; 290: 2277–2283.
9. Landrigan PJ, Trasande L, Thorpe LE, Gwynn C, Lioy
PJ, D’Alton ME, et al. The National Children’s Study: A 21-year
prospective study of 100 000 American children. Pediatrics 2006;
10. Ece A, Gürkan F, Kervancýoðlu M, Kocamaz H, Güneb
A, Atamer Y, el al. Oxidative stress, inflammation and early
cardiovascular damage in children with chronic renal failure. Pediatr
Neurol 2006: 21: 545-552.
11. Vukomirovic, D. Causes of deaths. In:
Statistical Yearbook of Serbia 2005, Statistical Office of the Republic of
Serbia, Belgrade, Serbia, 2005. p. 90.
12. Djordjevic D, Solevic T. Final report on
investi-gation of cause and degree of air pollution by harmful and
dangerous material on territory of Pancevo city. Ministry of Science and
Environ-mental Protection, Republic of Serbia. 2004. pp. 1-80. Available
from: URL: http://www.rnp.co.yu/rnp/ekologija/analiza_stanja_lat.jsp.
Accessed on December 14, 2007.
13. Hopkins WG. Estimating sample size for
magnitude-based inferences, Sportscience 2006; 10: 63-70.
14. Law About the Border Values, Methods of Measuring
Emission, Establishing Criteria for Measuring Sites and Recording Data
54/92, 30/99, 19/2006 (In Serbian Language). Ministry of Environment and
Spatial Planning. Available from: URL:http://www.ekoserb.sr.gov.yu/index.php?
option=com_remository&Itemid=89&func= startdown&id=614. Accessed on
November 29, 2007.
15. Friedewald WT, Levy RI, Fredrickson DS. Estimation
of the concentration of low-density lipoprotein cholesterol in plasma,
without use of preparative ultracentrifuge. Clin Chem 1972; 18:
16. Enas EA, Dhawan J, Petkar S. Coronary artery
disease in Asian Indians: lessons learnt and the role of lipoprotein(a).
Indian Heart J 1997; 49: 24-34.
17. Das B, Daga MK, Gupta SK. Lipid Pentad Index: A
novel bioindex for evaluation of lipid risk factors for atherosclerosis in
young adolescents and children of premature coronary artery disease
patients in India. Clin Biochem 2007; 40: 18-24.
18. Girotti MJ, Khan N, McLellan BA. Early measurement
of systemic lipid peroxidation products in plasma of major blunt trauma
patients. J Trauma 1991; 31: 32-35.
19. Auclair C, Voisin E. Nitroblue tetrazolium
reduction. In: Greenwald RA, editor. CRC Handbook of Methods for Oxygen
Radical Research. Boca Raton, Florida: CRC Press; 1985. pp. 123-132.
20. Misra HP, Fridovich I. The role of superoxide anion
in the autooxidation of epinephrine and a simple assay for superoxide
dismutase. J Biol Chem 1972; 247: 3170-3175.
21. Jiang ZY, Wollard ACS, Wolff SP. Lipid
hydroperoxides measurement of Xylenol Orange, comparison with the TBA
assay and iodometric method. Lipids 1991; 26: 853-856.
22. Witko-Sarsat V, Friedlander M, Capeille‘re-Blandin
C Nguyen-Khoa T, Nguyen AT, Zingraff J, et al. Advanced oxidation
protein products as a novel marker of oxidative stress in uremia. Kidney
Int 1996; 49: 1304.
23. Richter R, Furlong CE. Determination of
para-oxonase (pon1) status requires more than geno-typing.
Pharmacogenetics 1999; 9: 745-753.
24. Kotur-Stevuljevic J, Spasic S, Stefanovic A,
Zeljkovic A, Bogavac-Stanojevic N, Kalimanovska-Ostric D, et al.
Paraoxonase-1 (PON1) activity, but not PON1Q192R
phenotype, is a predictor of coronary artery disease in a middle-aged
Serbian population. Clin Chem Lab Med 2006; 44: 1106-1113.
25. Routledge HC, Ayres JG. Air pollution and the
heart. Occup Med 2005; 55: 439-447.
26. Nielsen F, Mikkelsen BB, Nielsen JB, Andersen HR,
Grandjean P. Plasma-malondialdehyde as biomarker for oxidative stress:
reference interval and effects of life-style factors. Clin Chem 1997; 43:
27. Del Rio D, Stewart AJ, Pellegrini N. A review of
recent studies on malondialdehyde as toxic molecule and biological marker
of oxidative stress. Nutr Metab Cardiovasc Dis 2005; 15: 316-328.
28. Zalata A, Yahia S, El-Bakary A, Elsheikha HA.
Increased DNA damage in children caused by passive smoking as assessed by
comet assay and oxidative stress. Mutat Res 2007; 629: 140-147.
29. Stocker R, Keaney JF. Role of Oxidative
Modifications in Atherosclerosis. Physiol Rev 2004; 84: 1381-1478.
30. Witko-Sarsat V, Friedlander M, Khoa TN,
Capeille‘re-Blandin C, Nguyen AT, Canteloup S, et al. Advanced
oxidation protein products as novel mediators of inflammation and monocyte
activation in chronic renal failure. J Immunol 1998; 161: 2524-2532.
31. Durrington PN, Mackness MJ. Paraoxonase and
atherosclerosis. Arterioscler Thromb Vasc Biol 2001; 21:
32. Aviram M, Hardak E, Vaya J, Mahmood S, Milo S,
Hoffman A, et al. Human serum paraoxonases (PON1) Q and R
selectively decrease lipid peroxides in human coronary and carotid
atherosclerotic lesions: PON1 esterase and peroxidase-like activities.
Circulation 2000; 101: 2510-2517.
33. Draganov DI, Teiber JF, Speelman A, Osawa Y,
Sunahara R, La Du BN, et al. Human paraoxonases (PON1, PON2 and
PON3) are lactonases with overlapping and distinct substrate
specificities. J Lipid Res 2005; 46: 1239-1247.
34. Executive summary of the Third Report of The
National Cholesterol Education Program (NCEP) Expert Panel on Detection,
Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult
Treatment Panel III). JAMA 2001; 285: 2486-2497.