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Increased levels of 8-hydroxy-2′-deoxyguanosine are attributable to organophosphate pesticide exposure among young children
Environmental Pollution 167 (2012) 110e114
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Increased levels of 8-hydroxy-20 -deoxyguanosine are attributable to organophosphate pesticide exposure among young children Guodong Ding a, b, Song Han c, Pei Wang d, Yu Gao e, Rong Shi e, Guoquan Wang f, Ying Tian a, e, * a
MOE and Shanghai Key Laboratory of Children’s Environmental Health, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China Department of Pediatrics, No. 3 People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China c Department of Epidemiology, Shenyang Medical College, Shenyang, China d Shanghai Pudong Center for Disease Control and Prevention, Shanghai, China e Department of Environmental Health, Shanghai Jiao Tong University School of Medicine, Shanghai, China f Shanghai Municipal Center for Disease Control and Prevention, Shanghai, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 September 2011 Received in revised form 5 April 2012 Accepted 8 April 2012
Oxidative damage has been proposed as an important mechanism linking pesticide exposure to health effects. A study of 268 young Shanghai children was conducted to examine the relationship between organophosphate pesticide (OP) exposure and a biomarker of oxidative DNA damage. Urine samples were analyzed for five nonspecific dialkyl phosphate (DAP) metabolites [dimethyl phosphates (DMs) and diethyl phosphates (DEs)] and 8-hydroxy-20 -deoxyguanosine (8-OHdG). The creatinine-adjusted median of 8-OHdG in urine samples was 3.99 ng/mg. Increased exposure to OPs was associated with greater levels of urinary 8-OHdG [total DAPs: ß (adjusted) ¼ 0.46 per log10 unit increase, 95% confidence interval (CI) ¼ 0.40e0.53, p ¼ 0.000; DMs: ß (adjusted) ¼ 0.34, 95% CI ¼ 0.28e0.41, p ¼ 0.000; DEs: ß (adjusted) ¼ 0.48, 95% CI ¼ 0.42e0.54, p ¼ 0.000]. Thus, the 8-OHdG biomarker is useful for increasing our understanding of the link between childhood exposure to OPs and health outcomes. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Organophosphate pesticide Oxidative stress 8-Hydroxy-20 -deoxyguanosine Children China
1. Introduction Organophosphate pesticides (OPs) are among the most widely used pesticides in the world, and they are deliberately released into both agricultural and residential settings to control pests. More than 300,000 tons of pesticides are used in agriculture each year in China, with OPs accounting for more than one-third of that total (Agriculture Information Network, 2006). Recent studies indicate that pesticide exposure is widespread and presents potential risks to humans, especially to susceptible populations such as pregnant women and children (Whyatt et al., 2004; Eskenazi et al., 2004). The potential adverse effects of pesticide exposure to children’s health, including reproductive outcomes, childhood cancers, neurobehavioral toxicity, and endocrine disruption have been well established (Garry, 2004). However, the molecular damage that results from the exposure to pesticides is less understood. Oxidative damage has been proposed frequently as an important
Abbreviations: 8-OHdG, 8-hydroxy-20 -deoxyguanosine; DMP, dimethyl phosphate; DMTP, dimethyl thiophosphate; DEP, diethyl phosphate; DETP, diethyl thiophosphate; DEDTP, diethyl dithiophosphate; DM, dimethyl phosphate; DE, diethyl phosphate; DAP, dialkyl phosphate; OP, organophosphate pesticide. * Corresponding author. E-mail address: [email protected] (Y. Tian). 0269-7491/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2012.04.001
mechanism that could link pesticide exposure to a number of health outcomes (Bagchi et al., 1995; Banerjee et al., 2001; Halliwell, 2002; Muniz et al., 2008). Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and the body’s ability to detoxify the reactive intermediates or easily repair the resulting damage. The most important form of ROS-induced biological toxicity is associated with damage to basic biomolecules, including proteins, lipids, and DNA (Valavanidis et al., 2009). There is compelling evidence from both whole animal and tissue culture studies that pesticides, especially OPs, increase ROS production and induce oxidative stress (Bagchi et al., 1995; Lodovici et al., 1997; Crumpton et al., 2000; Muniz et al., 2008). Increased levels of lipid peroxides in serum and urine as well as altered levels of glutathione (GSH) and antioxidant enzymes in the blood have been detected in several cases of pesticide poisoning (Banerjee et al., 1999; B1asiak et al., 1999; Muniz et al., 2008). Moreover, DNA damage comparable to that observed with oxidative stress has been detected following pesticide exposure (Lodovici et al., 1997; Shah et al., 1997; Lebailly et al., 1998; Lieberman et al., 1998; Muniz et al., 2008). Together, these factors indicate that oxidative damage may be an important mechanism by which OPs operate. Different types of oxidative DNA damage exist, but 8-hydroxy20 -deoxyguanosine (8-OHdG) is one of the most commonly
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observed base modifications known to occur; moreover, 8-OHdG has gained much attention because it is premutagenic, causing G-to-T transversions (Cheng et al., 1992) that may lead to mutagenesis. This mechanism of mutation suggests that nucleotide excision repair is essential for the elimination of 8-OHdG in humans. The specific process used to repair oxidative DNA damage results in the excised 8-OHdG adducts being excreted into the urine (Shigenaga et al., 1989; Marnett, 2000), and the amount of 8-OHdG in the urine is thought to represent the extent of whole body oxidative DNA damage (Pilger and Rüdiger, 2006). Thus, urinary 8-OHdG is regarded as a reliable biomarker for systemic oxidative DNA damage. Recently, the analysis of 8-OHdG in urine has been widely used to assess the extent of oxidative DNA damage caused by environmental chemicals, including OPs (Tope and Panemangalore, 2007; Muniz et al., 2008; Atherton et al., 2009). However, these investigations are often limited by the type of population studied, with most populations consisting of occupational adult workers (e.g., farmers, pesticide applicators, and floriculturists). Until now, little information has been available on the relationship between OP exposure in the general population and urinary 8-OHdG levels, especially in healthy children, who were the focus of our current research. To our knowledge, this study is the first to investigate urinary levels of OP metabolites and 8-OHdG in young children living in the metropolitan Shanghai area and the first to analyze the possible correlation between OP exposure and levels of oxidative stress. 2. Materials and methods 2.1. Participants and recruitment From February through October 2008, we recruited young children 2 years of age from the departments of child and adolescent healthcare from two Shanghai community hospitals. The participants were healthy children who were attending the departments for routine physical check-ups. Children considered eligible for the study were between 23 and 25 months of age and in general good health without current illness (cold or fever). No intrauterine distress, pathological jaundice, intrauterine infection, intracranial infection, or congenital disease was reported by the mothers. Overall, 310 children met the eligibility criteria, of whom 268 had parents who provided consent for their children to participate in this study (a response rate of 86.5%). All parents signed a consent form approved by the Shanghai Jiao Tong University School of Medicine Institutional Review Board. 2.2. Maternal interviews and assessments A trained research interviewer administered a 20-minute questionnaire to the mothers of the participants. The questionnaire included the following: demographic information and exposure characteristics, such as the child’s name, sex, age, illnesses and hospitalizations, and place of residence; environmental exposures (nearby field and para-occupational exposures); and child characteristics (dietary habits, handto-mouth contact, and passive smoking). Other relevant covariate information such as date of birth, weight, and height were derived from the children’s medical records. Socioeconomic information related to household income and parents’ education level was collected as well. 2.3. Urine collection Urine samples were collected from each participant during the study period. Research assistants collected the spot urine samples mid-morning through early afternoon using sterile urine cups. Following collection, the specimens were immediately aliquoted into pre-cleaned, bar-coded glass containers with Teflonlined caps, which were then stored at cold temperatures (2e4 C) or frozen until shipment. 2.4. Measurement of OP metabolite levels in urine Urine samples were shipped overnight on dry ice to the Shanghai Municipal Center for Disease Control & Prevention (CDC, Shanghai, China) and stored at 70 C until analysis. Frozen urine samples were thawed and analyzed for the presence of DAP metabolites using gas chromatography with flame photometric detection (GC-FPD). Five analytes were measured for each sample: DMP, DMTP, DEP, DETP, and DEDTP. To provide overall assessments of precision, accuracy, and the overall
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reliability of the method, we analyzed quality control (QC) samples along with the collected samples. QC samples were prepared as blank samples and inserted blindly among the study samples. The limit of detection (LOD) for each metabolite was calculated from the instrument response factor corresponding to a concentration having a peak area three times the baseline noise (blank signal). The specific LODs for the five metabolites were 2.0 mg/L for DMP and 1.0 mg/L for DMTP, DEP, DETP, and DEDTP. Individual metabolite levels below the LOD were assigned a value equal to the LOD divided by the square root of two (Hornung and Reed, 1990), and this value was included in each sum. Summed molar concentrations of the two dimethyl metabolites (DMP and DMTP) and the three diethyl metabolites (DEP, DETP, and DEDTP) were calculated to provide summary measures of exposure that were less affected by results below the LOD for individual metabolites. The conversion formula for each metabolite from its untransformed concentration (mg/L) to the corresponding molar concentration (nmol/L) has been described elsewhere (Arcury et al., 2006). The total DAP level was defined as the sum of the molar concentrations of the five metabolites. Metabolite concentrations were adjusted using creatinine concentrations to correct for variable urine dilutions in the spot urine samples. Creatinine concentrations in urine were determined using a commercially available diagnostic enzyme method (Vitros CREA slides, Ortho Clinical Diagnostics, Raritan, NJ). 2.5. Determination of urinary 8-OHdG levels Before examination, frozen urine samples were thawed and then centrifuged at 2000 g for 10 min to remove any suspended cell debris. The 8-OHdG levels in the supernatants were determined using a competitive enzyme-linked immunosorbent assay kit (ELISA, Japan Institute for the Control of Aging, Fukuroi, Japan) with a detection range of 0.5e200 ng/mL. Briefly, 50 mL of 8-OHdG monoclonal antibody N45.1 and the urine samples were loaded onto a microtiter plate pre-coated with 8-OHdG, and the plate was incubated at 37 C for 60 min according to the manufacturer’s instructions. The wells were washed three times. A horseradish peroxidase-conjugated secondary antibody was added to the wells, and the plates were incubated at 37 C for 60 min. The wells were again washed three times, and the substrate containing 3,30 ,5,50 -tetramethylbenzidine was added. The plates were then incubated at room temperature for 15 min, resulting in color development where the color intensity was proportional to the amount of antibody bound in each well. The color reaction was terminated by the addition of stop solution (1 M phosphoric acid), and the absorbance was measured using a computer-controlled spectrophotometric plate reader at a wavelength of 450 nm. The concentration of 8-OHdG in the test samples was interpolated from a standard curve drawn with the assistance of logarithmic transformation. Urinary 8-OHdG levels were subsequently adjusted on the basis of urinary creatinine levels (Wong et al., 2005). 2.6. Statistical analysis Because of the positively skewed distribution of the urinary 8-OHdG levels, we used nonparametric testing to test the differences between the urinary 8-OHdG levels for each variable. Median values of urinary DM, DE, and total DAP levels were used as cutoffs. We constructed multiple linear regression models to estimate the relationship between child OP exposure and levels of urinary 8-OHdG, adjusting for potential covariates. Passive smoking, parental education, and household income were included in the final model to be consistent with other studies. All regression analyses were run using log10-transformed creatinine-adjusted levels. Significance was based on a two-tailed test, and a p < 0.05 was considered significant.
3. Results In total, 268 children (150 boys and 118 girls) were involved in this study. Their mean age was 24.23 months (SD ¼ 0.32), their mean body weight was 12.87 kg (SD ¼ 1.47), and their mean body height was 88.69 cm (SD ¼ 6.68). Nearly all of the children (n ¼ 264, 98.5%) came from urban areas, and almost three in five (n ¼ 156, 58.2%) lived adjacent to a green park or agricultural field. More than 80% of the children consumed fresh fruit or vegetables daily. Approximately 15% of the children had frequent passive smoking exposure, but most children (approximately 60%) lived without exposure to smoking. Nearly 40% of these children displayed extensive hand-to-mouth contact. The majority of the mothers (89.2%) and fathers (95.5%) had completed high school or college. More than two-thirds of the children lived in households with a monthly income greater than RMB 5000. We did not adjust for para-occupation, a potential source of OP exposure, because only one father reported performing farm work.
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The OP urinary metabolite levels of the study sample, both unadjusted and adjusted for creatinine, are summarized in Table 1. The median urinary levels without creatinine adjustment were 18.26 nmol/L for DMs, 36.85 nmol/L for DEs, and 75.67 nmol/L for total DAPs. The creatinine-adjusted median urinary levels were 146.08 nmol/g for DMs, 167.68 nmol/g for DEs, and 369.06 nmol/g for total DAPs. Among the 268 urine samples, the frequencies of detection for DMP, DMTP, DEP, DETP, and DEDTP were 40.7% (n ¼ 109), 35.4% (n ¼ 95), 71.6% (n ¼ 192), 67.5% (n ¼ 181), and 2.6% (n ¼ 7), respectively. The median urinary 8-OHdG level for the study subjects was 3.99 ng/mg of creatinine (range 0.92e21.64 ng/mg of creatinine). No significant differences in urinary 8-OHdG levels were found in terms of gender, hand-to-mouth contact, dietary habits, passive smoking status, parental education, household income, or place of residence. However, we observed significant differences in urinary 8-OHdG levels when using the medians (nmol/g creatinine) of the urinary OP metabolite levels as cutoffs (high/low). Children with high urinary OP metabolite levels had much higher 8-OHdG levels than did those with low urinary OP metabolite levels (total DAPs: Z ¼ 9.656, p ¼ 0.000; DMs: Z ¼ 8.690, p ¼ 0.000; DEs: Z ¼ 9.380, p ¼ 0.000; ManneWhitney U test) (Table 2). Our univariate analysis showed that only the urinary OP metabolite levels (total DAPs, DMs, and DEs) were associated with elevated levels of urinary 8-OHdG (p < 0.01). Therefore, we performed regression models to assess the possible relationship of child OP exposure with urinary 8-OHdG levels after adjusting for passive smoking, parental education, and household income. Increased exposure to OPs was associated with greater levels of urinary 8-OHdG [total DAPs: ß (adjusted) ¼ 0.46 per log10 unit increase, 95% confidence interval (CI) ¼ 0.40e0.53, p ¼ 0.000; DMs: ß (adjusted) ¼ 0.34, 95% CI ¼ 0.28e0.41, p ¼ 0.000; DEs: ß (adjusted) ¼ 0.48, 95% CI ¼ 0.42e0.54, p ¼ 0.000]. 4. Discussion OPs are used throughout China in large quantities, and concerns regarding the adverse health effects of OPs in children are increasing because of the OP exposure problems associated with rapid development and a large population (Ye et al., 2007). Oxidative damage has been frequently proposed as an important mechanism that could link pesticide exposure to a number of health outcomes (Bagchi et al., 1995; Banerjee et al., 2001; Halliwell, 2002). In the present study, we assessed the relationship between OP exposure in children and oxidative stress levels and found significantly positive relationships between urinary OP metabolites and 8-OHdG levels. Under normal physiological conditions, a balance exists between endogenous ROS production and antioxidative mechanisms. When this balance is disrupted, oxidative stress generates extensive oxidative DNA damage, which, in turn, contributes to adverse health outcomes such as carcinogenesis and neurological
Table 1 Urinary DAP metabolite levels (nmol/L urine and nmol/g creatinine) in the study sample. Metabolite
Geometric mean
DMs (nmol/L) DMs (nmol/g) DEs (nmol/L) DEs (nmol/g) Total DAPs (nmol/L) Total DAPs (nmol/g)
33.95 155.02 40.66 185.62 86.99 397.15
Percentile of distribution 25th
50th
75th
10.23 60.89 18.76 83.43 45.07 197.87
18.26 146.08 36.85 167.68 75.67 369.06
56.73 359.18 67.11 415.33 134.81 800.51
Table 2 Urinary 8-OHdG levels (ng/mg creatinine) stratified by different variables. Variable
No.
Mean SE
Median (range)
Total
268
5.18 0.25
3.99 (0.92e21.64)
Sex Boys Girls
150 118
5.19 0.32 5.17 0.40
4.01 (0.92e20.37) 3.62 (0.96e21.64)
Hand-to-mouth contact Never Occasional Often
135 7 126
5.22 0.34 5.41 0.72 5.12 0.42
3.99 (0.96e21.64) 4.51 (0.92e17.49) 3.65 (0.97e21.37)
Fresh fruit or vegetables consumed weekly 3 times 21 4.44 1.13 4e6 times 26 4.76 0.77 Daily 221 5.30 0.27
3.65 (0.92e17.83) 4.47 (1.07e17.49) 4.73 (1.01e21.64)
Passive smoking Never Occasional Often
158 73 37
4.87 0.29 5.43 0.54 5.98 0.81
3.70 (0.92e19.60) 4.29 (0.99e20.37) 4.80 (0.96e21.64)
Maternal education (years)
86 182
4.74 0.43 5.39 0.31
3.65 (0.96e20.37) 3.99 (0.92e21.64)
Paternal education (years)
79 189
4.61 0.44 5.42 0.31
3.63 (0.96e20.37) 4.02 (0.92e21.64)
Household income (RMB/month) <5000 5000e10,000 >10,000
92 100 76
4.51 0.36 5.20 0.41 5.96 0.54
3.39 (0.92e20.37) 3.70 (0.99e17.83) 4.41 (0.99e21.64)
156
5.19 0.33
4.01 (0.96e21.64)
Place of residence Adjacent to green park or agricultural field Not adjacent
112
5.17 0.38
3.59 (0.92e19.60)
Adjusted urinary DM levelsa High (146.08 nmol/g creatinine) Low (<146.08 nmol/g creatinine)
134 134
7.31 0.41 3.05 0.14
6.66 (1.21e21.64)** 2.82 (0.92e7.48)
Adjusted urinary DE levelsa High (167.68 nmol/g creatinine) Low (<167.68 nmol/g creatinine)
134 134
7.38 0.40 2.98 0.14
6.34 (1.08e21.64)** 2.63 (0.92e7.33)
Adjusted urinary total DAP levelsa High (369.06 nmol/g creatinine) Low (<369.06 nmol/g creatinine)
134 134
7.43 0.40 2.92 0.13
6.60 (1.21e21.64)** 2.62 (0.92e7.92)
**p < 0.01. a Cutpoints were determined according to medians of urinary creatinine-adjusted levels among all subjects.
disease. In a case-control study, Yang et al. (2009) found that the level of urinary 8-OHdG in children with acute leukemia was significantly elevated compared with that in normal controls (11.92 15.42 vs. 4.03 4.70 ng/mg of creatinine). Urinary 8-OHdG was also higher in children with acute leukemia who were less than 3 years old than in 3e15 year olds (20.86 21.75 vs. 8.09 9.65 ng/mg). However, oxidative DNA damage is not only associated with endogenous oxidative stress but also with environmental oxidative stressors such as various environmental pollutants, lifestyle factors and carcinogenic substances (Valavanidis et al., 2009). Globally, OPs are some of the most important environmental pollutants, especially in developing countries where they are heavily used. Previous epidemiological studies have shown significantly positive correlations between urinary OP metabolites and 8-OHdG levels in agricultural workers, suggesting that an increase in OP exposure is associated with a high level of oxidative stress (Tope and Panemangalore, 2007; Muniz et al., 2008; Atherton et al., 2009). However, such studies are often limited by the type of population studied, with most being limited to occupational adult workers.
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Until now, urinary 8-OHdG has not been used to detect the effects of environmental OP exposure in the general population, especially in susceptible children. Children appear uniquely vulnerable to environmental pollutants based on disproportionately heavy exposures and inherent biological susceptibilities (Landrigan et al., 1998), thus making children the focus of many exposure assessments. Our study is the first in China to analyze the correlation between OP exposure and levels of oxidative stress in children. The results show that the exposure of children to OPs is associated with the increased generation of 8-OHdG and thus confirms previous findings that focused solely on occupational factors. These results may therefore be important for increasing our understanding of the link between low-level OP exposure and a number of health effects. It is possible that factors other than OP exposure elevated oxidative stress levels and influenced the amount of DNA damage. Smoking has often, but not always, been identified as a confounder of oxidative stress and DNA damage (Zhu et al., 1999). In this study, we observed that children who had passive smoking status had higher 8-OHdG levels than those who lived without exposure to smoking, although this discrepancy in 8-OHdG levels was not statistically significant. Cigarette smoke contains ROS, and the association between smoking and urinary 8-OHdG has been previously reported (Loft and Poulsen, 1996). Parental levels of education or household income, both used as alternative indicators of socioeconomic status, may influence the patterns of potential OP exposure and health care among children (Wong et al., 2005). Surprisingly, we observed that the urinary 8-OHdG levels in children from families of high socioeconomic status were always higher than the levels in families of low socioeconomic status, although this result was also not statistically significant. One possible explanation for this finding is that children from families of high socioeconomic status eat more fresh fruit and vegetables that generally have higher levels of pesticides than other foods. Furthermore, young children often spend most of their time indoors. House dust and fungi are the major residential pollutants in the subtropical Shanghai area and may increase cellular oxidative stress (Li et al., 1994; Wong et al., 2005). It is therefore important to consider the effects that exposure to the residential environment may have on the level of oxidative DNA damage that occurs in children. However, we did not find an association between urinary 8-OHdG levels and residential factors such as place of residence and hand-to-mouth contact. In our study, these indicators were self-reported and therefore were subjective. Thus, they could have resulted in the misclassification of exposure that might have reduced the observed associations. To our knowledge, this study is the first in China to investigate the relationship between OP exposure and oxidative stress levels in children. However, it is possible that urinary 8-OHdG levels were overestimated by the ELISA because the 8-OHdG monoclonal antibody used in our ELISA has similar binding affinities for the oxidized free base 8-hydroxyguanine and the oxidized nucleoside 8-hydroxyguanosine (Yin et al., 1995). In addition, as in most studies on the effects of pesticide exposure, we only measured biomarker levels at a single point in time, thus limiting our ability to determine average cumulative doses. 5. Conclusions In summary, the exposure of children to OPs was associated with the increased generation of 8-OHdG, suggesting that exposure to OPs may play an important role in oxidative damage in children. Given the potential implication of these results to public health, additional longitudinal research should be conducted to evaluate the risks of oxidative stress in terms of health outcomes.
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Acknowledgments We acknowledge the Department of Environmental Health staff, students, community partners, and participants and families, without whom this study would have been impossible. This publication was made possible by research support provided by the Natural Science Foundation of China (Grant No. 30872086, 81172625, and 30972533). The authors declare they have no competing financial interests. References Agriculture Information Network, 2006. Analysis of pesticides demand in China. Plant Doctor 19, 16e18 (in Chinese). Arcury, T.A., Grzywacz, J.G., Davis, S.W., Barr, D.B., Quandt, S.A., 2006. Organophosphorus pesticide urinary metabolite levels of children in farmworker households in eastern North Carolina. Am. J. Ind. Med. 49, 751e760. Atherton, K.M., Williams, F.M., Egea González, F.J., Glass, R., Rushton, S., Blain, P.G., Mutch, E., 2009. DNA damage in horticultural farmers: a pilot study showing an association with organophosphate pesticide exposure. 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Yang, Y., Tian, Y., Yan, C., Jin, X., Tang, J., Shen, X., 2009. Determinants of urinary 8hydroxy-20 -deoxyguanosine in Chinese children with acute leukemia. Environ. Toxicol. 24, 446e452. Ye, X., Fu, H., Guidotti, T., 2007. Environmental exposure and children’s health in China. Arch. Environ. Occup. Health 62, 61e73. Yin, B., Whyatt, R.M., Perera, F.P., Randall, M.C., Cooper, T.B., Santella, R.M., 1995. Determination of 8-hydroxydeoxyguanosine by an immunoaffinity chromatography-monoclonal antibody- based ELISA. Free Radic. Biol. Med. 18, 1023e1032. Zhu, C.Q., Lam, T.H., Jiang, C.Q., Wei, B.X., Lou, X., Liu, W.W., Lao, X.Q., Chen, Y.H., 1999. Lymphocyte DNA damage in cigarette factory workers measured by the Comet assay. Mutat. Res. 444, 1e6.
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Increased levels of 8-hydroxy-20 -deoxyguanosine are attributable to organophosphate pesticide exposure among young children Guodong Ding a, b, Song Han c, Pei Wang d, Yu Gao e, Rong Shi e, Guoquan Wang f, Ying Tian a, e, * a
MOE and Shanghai Key Laboratory of Children’s Environmental Health, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China Department of Pediatrics, No. 3 People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China c Department of Epidemiology, Shenyang Medical College, Shenyang, China d Shanghai Pudong Center for Disease Control and Prevention, Shanghai, China e Department of Environmental Health, Shanghai Jiao Tong University School of Medicine, Shanghai, China f Shanghai Municipal Center for Disease Control and Prevention, Shanghai, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 September 2011 Received in revised form 5 April 2012 Accepted 8 April 2012
Oxidative damage has been proposed as an important mechanism linking pesticide exposure to health effects. A study of 268 young Shanghai children was conducted to examine the relationship between organophosphate pesticide (OP) exposure and a biomarker of oxidative DNA damage. Urine samples were analyzed for five nonspecific dialkyl phosphate (DAP) metabolites [dimethyl phosphates (DMs) and diethyl phosphates (DEs)] and 8-hydroxy-20 -deoxyguanosine (8-OHdG). The creatinine-adjusted median of 8-OHdG in urine samples was 3.99 ng/mg. Increased exposure to OPs was associated with greater levels of urinary 8-OHdG [total DAPs: ß (adjusted) ¼ 0.46 per log10 unit increase, 95% confidence interval (CI) ¼ 0.40e0.53, p ¼ 0.000; DMs: ß (adjusted) ¼ 0.34, 95% CI ¼ 0.28e0.41, p ¼ 0.000; DEs: ß (adjusted) ¼ 0.48, 95% CI ¼ 0.42e0.54, p ¼ 0.000]. Thus, the 8-OHdG biomarker is useful for increasing our understanding of the link between childhood exposure to OPs and health outcomes. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Organophosphate pesticide Oxidative stress 8-Hydroxy-20 -deoxyguanosine Children China
1. Introduction Organophosphate pesticides (OPs) are among the most widely used pesticides in the world, and they are deliberately released into both agricultural and residential settings to control pests. More than 300,000 tons of pesticides are used in agriculture each year in China, with OPs accounting for more than one-third of that total (Agriculture Information Network, 2006). Recent studies indicate that pesticide exposure is widespread and presents potential risks to humans, especially to susceptible populations such as pregnant women and children (Whyatt et al., 2004; Eskenazi et al., 2004). The potential adverse effects of pesticide exposure to children’s health, including reproductive outcomes, childhood cancers, neurobehavioral toxicity, and endocrine disruption have been well established (Garry, 2004). However, the molecular damage that results from the exposure to pesticides is less understood. Oxidative damage has been proposed frequently as an important
Abbreviations: 8-OHdG, 8-hydroxy-20 -deoxyguanosine; DMP, dimethyl phosphate; DMTP, dimethyl thiophosphate; DEP, diethyl phosphate; DETP, diethyl thiophosphate; DEDTP, diethyl dithiophosphate; DM, dimethyl phosphate; DE, diethyl phosphate; DAP, dialkyl phosphate; OP, organophosphate pesticide. * Corresponding author. E-mail address: [email protected] (Y. Tian). 0269-7491/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2012.04.001
mechanism that could link pesticide exposure to a number of health outcomes (Bagchi et al., 1995; Banerjee et al., 2001; Halliwell, 2002; Muniz et al., 2008). Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and the body’s ability to detoxify the reactive intermediates or easily repair the resulting damage. The most important form of ROS-induced biological toxicity is associated with damage to basic biomolecules, including proteins, lipids, and DNA (Valavanidis et al., 2009). There is compelling evidence from both whole animal and tissue culture studies that pesticides, especially OPs, increase ROS production and induce oxidative stress (Bagchi et al., 1995; Lodovici et al., 1997; Crumpton et al., 2000; Muniz et al., 2008). Increased levels of lipid peroxides in serum and urine as well as altered levels of glutathione (GSH) and antioxidant enzymes in the blood have been detected in several cases of pesticide poisoning (Banerjee et al., 1999; B1asiak et al., 1999; Muniz et al., 2008). Moreover, DNA damage comparable to that observed with oxidative stress has been detected following pesticide exposure (Lodovici et al., 1997; Shah et al., 1997; Lebailly et al., 1998; Lieberman et al., 1998; Muniz et al., 2008). Together, these factors indicate that oxidative damage may be an important mechanism by which OPs operate. Different types of oxidative DNA damage exist, but 8-hydroxy20 -deoxyguanosine (8-OHdG) is one of the most commonly
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observed base modifications known to occur; moreover, 8-OHdG has gained much attention because it is premutagenic, causing G-to-T transversions (Cheng et al., 1992) that may lead to mutagenesis. This mechanism of mutation suggests that nucleotide excision repair is essential for the elimination of 8-OHdG in humans. The specific process used to repair oxidative DNA damage results in the excised 8-OHdG adducts being excreted into the urine (Shigenaga et al., 1989; Marnett, 2000), and the amount of 8-OHdG in the urine is thought to represent the extent of whole body oxidative DNA damage (Pilger and Rüdiger, 2006). Thus, urinary 8-OHdG is regarded as a reliable biomarker for systemic oxidative DNA damage. Recently, the analysis of 8-OHdG in urine has been widely used to assess the extent of oxidative DNA damage caused by environmental chemicals, including OPs (Tope and Panemangalore, 2007; Muniz et al., 2008; Atherton et al., 2009). However, these investigations are often limited by the type of population studied, with most populations consisting of occupational adult workers (e.g., farmers, pesticide applicators, and floriculturists). Until now, little information has been available on the relationship between OP exposure in the general population and urinary 8-OHdG levels, especially in healthy children, who were the focus of our current research. To our knowledge, this study is the first to investigate urinary levels of OP metabolites and 8-OHdG in young children living in the metropolitan Shanghai area and the first to analyze the possible correlation between OP exposure and levels of oxidative stress. 2. Materials and methods 2.1. Participants and recruitment From February through October 2008, we recruited young children 2 years of age from the departments of child and adolescent healthcare from two Shanghai community hospitals. The participants were healthy children who were attending the departments for routine physical check-ups. Children considered eligible for the study were between 23 and 25 months of age and in general good health without current illness (cold or fever). No intrauterine distress, pathological jaundice, intrauterine infection, intracranial infection, or congenital disease was reported by the mothers. Overall, 310 children met the eligibility criteria, of whom 268 had parents who provided consent for their children to participate in this study (a response rate of 86.5%). All parents signed a consent form approved by the Shanghai Jiao Tong University School of Medicine Institutional Review Board. 2.2. Maternal interviews and assessments A trained research interviewer administered a 20-minute questionnaire to the mothers of the participants. The questionnaire included the following: demographic information and exposure characteristics, such as the child’s name, sex, age, illnesses and hospitalizations, and place of residence; environmental exposures (nearby field and para-occupational exposures); and child characteristics (dietary habits, handto-mouth contact, and passive smoking). Other relevant covariate information such as date of birth, weight, and height were derived from the children’s medical records. Socioeconomic information related to household income and parents’ education level was collected as well. 2.3. Urine collection Urine samples were collected from each participant during the study period. Research assistants collected the spot urine samples mid-morning through early afternoon using sterile urine cups. Following collection, the specimens were immediately aliquoted into pre-cleaned, bar-coded glass containers with Teflonlined caps, which were then stored at cold temperatures (2e4 C) or frozen until shipment. 2.4. Measurement of OP metabolite levels in urine Urine samples were shipped overnight on dry ice to the Shanghai Municipal Center for Disease Control & Prevention (CDC, Shanghai, China) and stored at 70 C until analysis. Frozen urine samples were thawed and analyzed for the presence of DAP metabolites using gas chromatography with flame photometric detection (GC-FPD). Five analytes were measured for each sample: DMP, DMTP, DEP, DETP, and DEDTP. To provide overall assessments of precision, accuracy, and the overall
111
reliability of the method, we analyzed quality control (QC) samples along with the collected samples. QC samples were prepared as blank samples and inserted blindly among the study samples. The limit of detection (LOD) for each metabolite was calculated from the instrument response factor corresponding to a concentration having a peak area three times the baseline noise (blank signal). The specific LODs for the five metabolites were 2.0 mg/L for DMP and 1.0 mg/L for DMTP, DEP, DETP, and DEDTP. Individual metabolite levels below the LOD were assigned a value equal to the LOD divided by the square root of two (Hornung and Reed, 1990), and this value was included in each sum. Summed molar concentrations of the two dimethyl metabolites (DMP and DMTP) and the three diethyl metabolites (DEP, DETP, and DEDTP) were calculated to provide summary measures of exposure that were less affected by results below the LOD for individual metabolites. The conversion formula for each metabolite from its untransformed concentration (mg/L) to the corresponding molar concentration (nmol/L) has been described elsewhere (Arcury et al., 2006). The total DAP level was defined as the sum of the molar concentrations of the five metabolites. Metabolite concentrations were adjusted using creatinine concentrations to correct for variable urine dilutions in the spot urine samples. Creatinine concentrations in urine were determined using a commercially available diagnostic enzyme method (Vitros CREA slides, Ortho Clinical Diagnostics, Raritan, NJ). 2.5. Determination of urinary 8-OHdG levels Before examination, frozen urine samples were thawed and then centrifuged at 2000 g for 10 min to remove any suspended cell debris. The 8-OHdG levels in the supernatants were determined using a competitive enzyme-linked immunosorbent assay kit (ELISA, Japan Institute for the Control of Aging, Fukuroi, Japan) with a detection range of 0.5e200 ng/mL. Briefly, 50 mL of 8-OHdG monoclonal antibody N45.1 and the urine samples were loaded onto a microtiter plate pre-coated with 8-OHdG, and the plate was incubated at 37 C for 60 min according to the manufacturer’s instructions. The wells were washed three times. A horseradish peroxidase-conjugated secondary antibody was added to the wells, and the plates were incubated at 37 C for 60 min. The wells were again washed three times, and the substrate containing 3,30 ,5,50 -tetramethylbenzidine was added. The plates were then incubated at room temperature for 15 min, resulting in color development where the color intensity was proportional to the amount of antibody bound in each well. The color reaction was terminated by the addition of stop solution (1 M phosphoric acid), and the absorbance was measured using a computer-controlled spectrophotometric plate reader at a wavelength of 450 nm. The concentration of 8-OHdG in the test samples was interpolated from a standard curve drawn with the assistance of logarithmic transformation. Urinary 8-OHdG levels were subsequently adjusted on the basis of urinary creatinine levels (Wong et al., 2005). 2.6. Statistical analysis Because of the positively skewed distribution of the urinary 8-OHdG levels, we used nonparametric testing to test the differences between the urinary 8-OHdG levels for each variable. Median values of urinary DM, DE, and total DAP levels were used as cutoffs. We constructed multiple linear regression models to estimate the relationship between child OP exposure and levels of urinary 8-OHdG, adjusting for potential covariates. Passive smoking, parental education, and household income were included in the final model to be consistent with other studies. All regression analyses were run using log10-transformed creatinine-adjusted levels. Significance was based on a two-tailed test, and a p < 0.05 was considered significant.
3. Results In total, 268 children (150 boys and 118 girls) were involved in this study. Their mean age was 24.23 months (SD ¼ 0.32), their mean body weight was 12.87 kg (SD ¼ 1.47), and their mean body height was 88.69 cm (SD ¼ 6.68). Nearly all of the children (n ¼ 264, 98.5%) came from urban areas, and almost three in five (n ¼ 156, 58.2%) lived adjacent to a green park or agricultural field. More than 80% of the children consumed fresh fruit or vegetables daily. Approximately 15% of the children had frequent passive smoking exposure, but most children (approximately 60%) lived without exposure to smoking. Nearly 40% of these children displayed extensive hand-to-mouth contact. The majority of the mothers (89.2%) and fathers (95.5%) had completed high school or college. More than two-thirds of the children lived in households with a monthly income greater than RMB 5000. We did not adjust for para-occupation, a potential source of OP exposure, because only one father reported performing farm work.
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The OP urinary metabolite levels of the study sample, both unadjusted and adjusted for creatinine, are summarized in Table 1. The median urinary levels without creatinine adjustment were 18.26 nmol/L for DMs, 36.85 nmol/L for DEs, and 75.67 nmol/L for total DAPs. The creatinine-adjusted median urinary levels were 146.08 nmol/g for DMs, 167.68 nmol/g for DEs, and 369.06 nmol/g for total DAPs. Among the 268 urine samples, the frequencies of detection for DMP, DMTP, DEP, DETP, and DEDTP were 40.7% (n ¼ 109), 35.4% (n ¼ 95), 71.6% (n ¼ 192), 67.5% (n ¼ 181), and 2.6% (n ¼ 7), respectively. The median urinary 8-OHdG level for the study subjects was 3.99 ng/mg of creatinine (range 0.92e21.64 ng/mg of creatinine). No significant differences in urinary 8-OHdG levels were found in terms of gender, hand-to-mouth contact, dietary habits, passive smoking status, parental education, household income, or place of residence. However, we observed significant differences in urinary 8-OHdG levels when using the medians (nmol/g creatinine) of the urinary OP metabolite levels as cutoffs (high/low). Children with high urinary OP metabolite levels had much higher 8-OHdG levels than did those with low urinary OP metabolite levels (total DAPs: Z ¼ 9.656, p ¼ 0.000; DMs: Z ¼ 8.690, p ¼ 0.000; DEs: Z ¼ 9.380, p ¼ 0.000; ManneWhitney U test) (Table 2). Our univariate analysis showed that only the urinary OP metabolite levels (total DAPs, DMs, and DEs) were associated with elevated levels of urinary 8-OHdG (p < 0.01). Therefore, we performed regression models to assess the possible relationship of child OP exposure with urinary 8-OHdG levels after adjusting for passive smoking, parental education, and household income. Increased exposure to OPs was associated with greater levels of urinary 8-OHdG [total DAPs: ß (adjusted) ¼ 0.46 per log10 unit increase, 95% confidence interval (CI) ¼ 0.40e0.53, p ¼ 0.000; DMs: ß (adjusted) ¼ 0.34, 95% CI ¼ 0.28e0.41, p ¼ 0.000; DEs: ß (adjusted) ¼ 0.48, 95% CI ¼ 0.42e0.54, p ¼ 0.000]. 4. Discussion OPs are used throughout China in large quantities, and concerns regarding the adverse health effects of OPs in children are increasing because of the OP exposure problems associated with rapid development and a large population (Ye et al., 2007). Oxidative damage has been frequently proposed as an important mechanism that could link pesticide exposure to a number of health outcomes (Bagchi et al., 1995; Banerjee et al., 2001; Halliwell, 2002). In the present study, we assessed the relationship between OP exposure in children and oxidative stress levels and found significantly positive relationships between urinary OP metabolites and 8-OHdG levels. Under normal physiological conditions, a balance exists between endogenous ROS production and antioxidative mechanisms. When this balance is disrupted, oxidative stress generates extensive oxidative DNA damage, which, in turn, contributes to adverse health outcomes such as carcinogenesis and neurological
Table 1 Urinary DAP metabolite levels (nmol/L urine and nmol/g creatinine) in the study sample. Metabolite
Geometric mean
DMs (nmol/L) DMs (nmol/g) DEs (nmol/L) DEs (nmol/g) Total DAPs (nmol/L) Total DAPs (nmol/g)
33.95 155.02 40.66 185.62 86.99 397.15
Percentile of distribution 25th
50th
75th
10.23 60.89 18.76 83.43 45.07 197.87
18.26 146.08 36.85 167.68 75.67 369.06
56.73 359.18 67.11 415.33 134.81 800.51
Table 2 Urinary 8-OHdG levels (ng/mg creatinine) stratified by different variables. Variable
No.
Mean SE
Median (range)
Total
268
5.18 0.25
3.99 (0.92e21.64)
Sex Boys Girls
150 118
5.19 0.32 5.17 0.40
4.01 (0.92e20.37) 3.62 (0.96e21.64)
Hand-to-mouth contact Never Occasional Often
135 7 126
5.22 0.34 5.41 0.72 5.12 0.42
3.99 (0.96e21.64) 4.51 (0.92e17.49) 3.65 (0.97e21.37)
Fresh fruit or vegetables consumed weekly 3 times 21 4.44 1.13 4e6 times 26 4.76 0.77 Daily 221 5.30 0.27
3.65 (0.92e17.83) 4.47 (1.07e17.49) 4.73 (1.01e21.64)
Passive smoking Never Occasional Often
158 73 37
4.87 0.29 5.43 0.54 5.98 0.81
3.70 (0.92e19.60) 4.29 (0.99e20.37) 4.80 (0.96e21.64)
Maternal education (years)
86 182
4.74 0.43 5.39 0.31
3.65 (0.96e20.37) 3.99 (0.92e21.64)
Paternal education (years)
79 189
4.61 0.44 5.42 0.31
3.63 (0.96e20.37) 4.02 (0.92e21.64)
Household income (RMB/month) <5000 5000e10,000 >10,000
92 100 76
4.51 0.36 5.20 0.41 5.96 0.54
3.39 (0.92e20.37) 3.70 (0.99e17.83) 4.41 (0.99e21.64)
156
5.19 0.33
4.01 (0.96e21.64)
Place of residence Adjacent to green park or agricultural field Not adjacent
112
5.17 0.38
3.59 (0.92e19.60)
Adjusted urinary DM levelsa High (146.08 nmol/g creatinine) Low (<146.08 nmol/g creatinine)
134 134
7.31 0.41 3.05 0.14
6.66 (1.21e21.64)** 2.82 (0.92e7.48)
Adjusted urinary DE levelsa High (167.68 nmol/g creatinine) Low (<167.68 nmol/g creatinine)
134 134
7.38 0.40 2.98 0.14
6.34 (1.08e21.64)** 2.63 (0.92e7.33)
Adjusted urinary total DAP levelsa High (369.06 nmol/g creatinine) Low (<369.06 nmol/g creatinine)
134 134
7.43 0.40 2.92 0.13
6.60 (1.21e21.64)** 2.62 (0.92e7.92)
**p < 0.01. a Cutpoints were determined according to medians of urinary creatinine-adjusted levels among all subjects.
disease. In a case-control study, Yang et al. (2009) found that the level of urinary 8-OHdG in children with acute leukemia was significantly elevated compared with that in normal controls (11.92 15.42 vs. 4.03 4.70 ng/mg of creatinine). Urinary 8-OHdG was also higher in children with acute leukemia who were less than 3 years old than in 3e15 year olds (20.86 21.75 vs. 8.09 9.65 ng/mg). However, oxidative DNA damage is not only associated with endogenous oxidative stress but also with environmental oxidative stressors such as various environmental pollutants, lifestyle factors and carcinogenic substances (Valavanidis et al., 2009). Globally, OPs are some of the most important environmental pollutants, especially in developing countries where they are heavily used. Previous epidemiological studies have shown significantly positive correlations between urinary OP metabolites and 8-OHdG levels in agricultural workers, suggesting that an increase in OP exposure is associated with a high level of oxidative stress (Tope and Panemangalore, 2007; Muniz et al., 2008; Atherton et al., 2009). However, such studies are often limited by the type of population studied, with most being limited to occupational adult workers.
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Until now, urinary 8-OHdG has not been used to detect the effects of environmental OP exposure in the general population, especially in susceptible children. Children appear uniquely vulnerable to environmental pollutants based on disproportionately heavy exposures and inherent biological susceptibilities (Landrigan et al., 1998), thus making children the focus of many exposure assessments. Our study is the first in China to analyze the correlation between OP exposure and levels of oxidative stress in children. The results show that the exposure of children to OPs is associated with the increased generation of 8-OHdG and thus confirms previous findings that focused solely on occupational factors. These results may therefore be important for increasing our understanding of the link between low-level OP exposure and a number of health effects. It is possible that factors other than OP exposure elevated oxidative stress levels and influenced the amount of DNA damage. Smoking has often, but not always, been identified as a confounder of oxidative stress and DNA damage (Zhu et al., 1999). In this study, we observed that children who had passive smoking status had higher 8-OHdG levels than those who lived without exposure to smoking, although this discrepancy in 8-OHdG levels was not statistically significant. Cigarette smoke contains ROS, and the association between smoking and urinary 8-OHdG has been previously reported (Loft and Poulsen, 1996). Parental levels of education or household income, both used as alternative indicators of socioeconomic status, may influence the patterns of potential OP exposure and health care among children (Wong et al., 2005). Surprisingly, we observed that the urinary 8-OHdG levels in children from families of high socioeconomic status were always higher than the levels in families of low socioeconomic status, although this result was also not statistically significant. One possible explanation for this finding is that children from families of high socioeconomic status eat more fresh fruit and vegetables that generally have higher levels of pesticides than other foods. Furthermore, young children often spend most of their time indoors. House dust and fungi are the major residential pollutants in the subtropical Shanghai area and may increase cellular oxidative stress (Li et al., 1994; Wong et al., 2005). It is therefore important to consider the effects that exposure to the residential environment may have on the level of oxidative DNA damage that occurs in children. However, we did not find an association between urinary 8-OHdG levels and residential factors such as place of residence and hand-to-mouth contact. In our study, these indicators were self-reported and therefore were subjective. Thus, they could have resulted in the misclassification of exposure that might have reduced the observed associations. To our knowledge, this study is the first in China to investigate the relationship between OP exposure and oxidative stress levels in children. However, it is possible that urinary 8-OHdG levels were overestimated by the ELISA because the 8-OHdG monoclonal antibody used in our ELISA has similar binding affinities for the oxidized free base 8-hydroxyguanine and the oxidized nucleoside 8-hydroxyguanosine (Yin et al., 1995). In addition, as in most studies on the effects of pesticide exposure, we only measured biomarker levels at a single point in time, thus limiting our ability to determine average cumulative doses. 5. Conclusions In summary, the exposure of children to OPs was associated with the increased generation of 8-OHdG, suggesting that exposure to OPs may play an important role in oxidative damage in children. Given the potential implication of these results to public health, additional longitudinal research should be conducted to evaluate the risks of oxidative stress in terms of health outcomes.
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Acknowledgments We acknowledge the Department of Environmental Health staff, students, community partners, and participants and families, without whom this study would have been impossible. This publication was made possible by research support provided by the Natural Science Foundation of China (Grant No. 30872086, 81172625, and 30972533). The authors declare they have no competing financial interests. References Agriculture Information Network, 2006. Analysis of pesticides demand in China. Plant Doctor 19, 16e18 (in Chinese). Arcury, T.A., Grzywacz, J.G., Davis, S.W., Barr, D.B., Quandt, S.A., 2006. Organophosphorus pesticide urinary metabolite levels of children in farmworker households in eastern North Carolina. Am. J. Ind. Med. 49, 751e760. Atherton, K.M., Williams, F.M., Egea González, F.J., Glass, R., Rushton, S., Blain, P.G., Mutch, E., 2009. DNA damage in horticultural farmers: a pilot study showing an association with organophosphate pesticide exposure. 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