Oxidative Stress and Anti-oxidative Defense in Schoolchildren Residing in a Petrochemical Industry Environment
 

Received: August 22, 2008;
Initial review: October 3, 2008;
Accepted: February 27, 2009.

Published online 2009 May 20.
PII:S097475590800522-1
 

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.

Many 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 schoolchildren.

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 protocol.

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).

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 PON1₁₉₂ activity 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 activity ratio(24).

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.

TABLE I 



Basic Demographic Characteristics and Biochemical Parameters of the Study Groups 

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).

TABLE II 



Oxidative Stress Status and Anti-Oxidant Defense Parameters in the Study Groups

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 (QQ_EG vs. QQ_NEG 0.49 vs. 0.47; QR_EG vs. QR_NEG 0.42 vs. 0.43 and RR_EG vs RR_NEG 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).

TABLE III 



PON1 Activity Toward Paraoxon and Diazoxon According to PON1 Activity Phenotypes 

POase, PON1 activity towards paraoxon; DZOase, PON1 activity towards diazoxon; QQ, QR,RR, 

phenotype subgroups for PON1192 isoenzyme.

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).

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 PON1_192RR isoform is less capable for antioxidative protection than PON1_192QR or PON1_192QQ 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.

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.