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Maternal Arsenic Exposure and DNA Damage Biomarkers, and the Associations with Birth Outcomes in a General Population from Taiwan

  • Wei-Chun Chou,

    Affiliations Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan, Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli County, Taiwan

  • Yu-The Chung,

    Affiliation Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli County, Taiwan

  • Hsiao-Yen Chen,

    Affiliation Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli County, Taiwan

  • Chien-Jen Wang,

    Affiliation Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli County, Taiwan

  • Tsung-Ho Ying,

    Affiliation Department of Obstetrics and Gynecology, Chung Shan Medical University Hospital, Taichung, Taiwan

  • Chun-Yu Chuang , (CC); (SW)

    Affiliation Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan

  • Ying-Chih Tseng ,

    Contributed equally to this work with: Ying-Chih Tseng, Shu-Li Wang

    Affiliation Department of Obstetrics and Gynecology, Hsinchu Cathay General Hospital, Hsinchu, Taiwan

  • Shu-Li Wang

    Contributed equally to this work with: Ying-Chih Tseng, Shu-Li Wang (CC); (SW)

    Affiliations Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli County, Taiwan, Epidemiology Branch, National Institutes and Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, United States of America

Maternal Arsenic Exposure and DNA Damage Biomarkers, and the Associations with Birth Outcomes in a General Population from Taiwan

  • Wei-Chun Chou, 
  • Yu-The Chung, 
  • Hsiao-Yen Chen, 
  • Chien-Jen Wang, 
  • Tsung-Ho Ying, 
  • Chun-Yu Chuang, 
  • Ying-Chih Tseng, 
  • Shu-Li Wang


Inorganic arsenic (iAs) is an established transplacental agent known to affect fetal development in animal studies. However, iAs has not been adequately studied in the general population with respect to iAs exposure during pregnancy and its impact on the health status of newborns. The aims of this study were to 1) elucidate the association between arsenic exposure and oxidative/methylated DNA damage in pregnant women, and 2) determine the association with birth outcomes. A birth cohort study of 299 pregnant mother-newborn pairs was recruited during 2001–2002 in Taiwan. We collected maternal urine samples during the 3rd trimester for measuring iAs and its metabolites. We used high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC-ICP-MS) for quantifications of the arsenic species. Liquid chromatography/tandem mass spectrometer (LC-MS/MS) was used to measure the 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and N7-methylguanosine (N7-MeG) DNA damage biomarkers. Birth outcomes were collected to assess the associations with maternal arsenic exposure and the DNA damage biomarkers. Multiple regression analyses showed that maternal urinary iAs had positive associations with the methylated N7-MeG (beta  = 0.35, p<0.001) and oxidative 8-oxodG (beta  = 0.24, p<0.001) DNA damage biomarkers, and a decreased one-minute (1-min) Apgar score (beta  = -0.23, p = 0.041). Maternal N7-MeG was also associated with a decreased 1-min Apgar score (beta  = −0.25, p = 0.042). Mutual adjustment for iAs and N7-MeG showed an independent and significant prediction for a decreased 1-min Apgar score of iAs (beta  = −0.28, p = 0.036). Maternal iAs exposure was associated with both maternal DNA damage and adverse newborn health. Maternal N7-MeG levels might be a novel biomarker for monitoring fetal health related to iAs.


Health status at birth is an important determinant of morbidity and mortality in early childhood [1] and of chronic disease in adulthood [2]. For example, birth weight is not only reflective of maternal health status, but also predictive of the probability for newborn survival, development, and long-term health [3]. The Apgar score is a routine for evaluating the physical condition of the newborn, including heart rate, respiratory effort, muscle tone, reflex irritability, and skin color shortly after delivery. A score ≥7 indicates that the condition of the newborn is good-to-excellent [4]. Otherwise, immediate extra medical care or even an intensive care unit admission would be necessary. Newborns with low birth weight (LBW) or low Apgar scores often develop various negative health consequences. Long-term effects of LBW include increased risk of cardiovascular disease, type 2 diabetes mellitus, and impaired reproductive function [5]. Several studies have shown that low Apgar scores may be associated with an increased risk of reduced cognitive function and increased learning difficulties later in life [6]. Arsenic is a well-known toxicant and carcinogen, and increasing evidence indicates that arsenic may adversely affect pregnancy outcomes and development of the newborn.

Arsenic easily crosses the placenta [7], and even moderate exposure to arsenic during pregnancy has been associated with adverse health outcomes in the fetus [8]. Studies have shown that prenatal arsenic exposure is inversely associated with birth weight in Bangladesh [9], [10], and Inner Mongolia [11]. Prenatal arsenic exposure at low-to-moderate levels might also have effects on the fetus, but more evidence is needed [12].

Disrupted placentation [13] and endocrine disturbance [14] have been reported for arsenic-related adverse pregnancy outcomes. However, the mechanisms require further investigation. Studies of mice have reported that arsenic leads to an increase in oxidative stress and a subsequent increase in 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in tissues of tested animals [15], [16]. Increased urinary 8-oxodG in pregnant women has been linked to arsenic exposure [17]. Recently, maternal DNA damage mediated by arsenic exposure has been proposed as one mechanism responsible for fetal programming [18]. There is, however, little information on the formation of methylated DNA damage induced by arsenic exposure. Arsenic-treated mice have an arsenic-related increase in hepatic N7-methylguanine (N7-MeG), a marker of methylated DNA damage that reflects the overall rate of DNA methylation [19], [20], but this finding has not been confirmed in humans.

We sought to determine whether prenatal exposure to low-to-moderate levels of arsenic is associated with maternal oxidative/methylated DNA damage, and to evaluate the associations with birth outcomes. We found decreased Apgar score was associated prenatal arsenic exposure, and the maternal methylated DNA damage as the biomarker in a general population from central Taiwan.


Ethics statement

The Human Ethical Committee of the National Health Research Institutes in Taiwan approved this study. Before participating in the study, all pregnant women signed informed consent forms after receiving detailed explanations of the benefits and risks.

Study participants

There is no record of high arsenic exposure in this study region; and, a representative sample for the general population was targeted. Subject recruitment [21] and preliminary results [22] were described previously. Briefly, all pregnant women who received care at the local medical center serving the general population were invited to join our study between December 2000 and November 2001 (Figure 1). The pregnant women were generally healthy and living in the local area with good prenatal appointment compliance. Initially, 430 of 610 pregnant women were volunteers recruited at 8 weeks gestation on average. The subjects provided urine samples during the third trimester (28–38 weeks gestation). Of the 430 pregnant women, 107 did not have sufficient data or provide urine, and 10 women who smoked cigarettes were excluded. To facilitate compliance with an independent outcomes assumption, one of the two subjects was randomly selected for the nine pairs of twins. In addition, we had missing data on 5 newborn heath assessments due to loss to follow-up. Consequently, a total of 299 mother-newborn pairs were reported.

Determination of arsenic metabolites

The pregnant women collected urine in a 200-ml paper cup. The research assistant then allocated the urine specimens to 10-ml polypropylene tubes and stored them in a −20°C freezer until analysis. The concentrations of arsenite (As3+), arsenate (As5+), monomethylarsonic acid (MMA), and dimethyl arsenate (DMA) in the urine samples were measured using high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC-ICP-MS). We used anion exchange columns (Hamilton PRP X-100 [10 µm particle size, 250 mm×4.1 mm]) for arsenic speciation. The mobile phase was 100 mmol/ L Na2HPO4/ NaH2PO4 (pH = 5.75) at a flow rate of 1 mL/min. The limitations of detection (LOD) for the various species were 0.09 µg/L for As3+, 0.05 µg/L for As5+, 0.05 µg/L for MMA, and 0.04 µg/L for DMA.

Blanks were analyzed every 10 samples and the results were <0.01 µg/L of arsenic. Five standards spanning the range of urinary arsenic concentrations were used to set the calibration curve in each run of 10 samples. The analysis of samples proceeded only when the correlation coefficients of the standard curves with outcomes were >0.999. Duplicates were run every 10 samples and the coefficients of variance (CV %) were <5%. The recovery rate was between 90% and 110% with standard addition relevant to samples ranging from 2–5 µg/L for all arsenic species. The interbatch CVs were <10% in all samples ranging from 4.9%–7.8%. The inter-lab comparisons were conducted by participating in the Round Table Program organized by Recipe Chemicals and Instruments GmbH (Munich, Germany). We fulfilled the reference values for all tested samples.

Creatinine was measured by the Beckman Synchron LX20 auto-system (Beckman Coulter, Brea, CA, USA) in the central lab of Chung-Ho Memorial Hospital of Kaohsiung Medical University using a spectrophotometric method with picric acid as the reactive at 520 nm. We used total arsenic (tAs) as the sum of inorganic arsenic (As3+ and As5+) and organic arsenic (MMA and DMA) divided by the urinary creatinine in the subsequent analysis [23].

DNA damage biomarker quantification

Urinary 8-oxodG and N7-MeG were determined using liquid chromatography/tandem mass spectrometry (LC-MS/MS). Urine samples were thawed at room temperature and centrifuged at 10000 g for 5 min. Then, 20 µL samples of urine were diluted 20 times with 96% acetonitrile containing 0.1% formic acid (FA; 380 µL). An 80-µL aliquot of diluted urine was spiked with 20 µL of a mixed solution consisting of 15N5-N7-MeG and 15N5-8-oxodG as an internal standard and then vortexed. A 100-µL sample of prepared urine was injected directly into the on-line solid-phase extraction column (Sep-Pak C18 cartridges, 1 g/6 mL; Waters, Milford, MA, USA). After automatic sample cleanup, LC-MS/MS analysis was done using a PE Series 200 HPLC system interfaced with a PE Sciex API 3000 triple quadruple mass spectrometer with an electrospray ion source [24] (Applied Biosystems, Toronto, Canada). The LOD of the 8-oxodG and N7-MeG was 0.01 µg/L and the coefficient of variation (CV) was 2.5% and 5.5% for the 8-oxo-dG and N7-MeG, respectively.

Birth outcomes and covariates

The physical parameters of the newborns, including gestational age, sex, birth weight and height, head and chest circumference, and Apgar score, were measured and recorded by the same pediatrician and a well-trained assistant. The nurses in the maternity ward measured birth weight using a scale for babies (Misaki baby scale, Japan) that was calibrated before each use. Calibration with a standard weight (5 kg) at regular intervals showed that all scales were stable and precise. Birth length was measured to the nearest 0.1 cm using a wooden length board, and the head and chest circumferences were measured using a metal measuring tape. The Apgar score was determined for each baby to ascertain physical condition and determine the immediate need for extra medical or emergency care. A pediatrician or trained nurse assigned Apgar scores based on breathing effort, heart rate, muscle tone, reflexes, and skin color. Each category was scored with a 0, 1, or 2, according to the observed conditions. The Apgar test was given to babies at 1 and 5 minutes [25].

In the current study, adverse birth outcomes in newborns involved a low birth weight and low Apgar scores. We suggest that birth length might be correlated with the physical condition of the baby, such as body relaxation, and head and chest girth with the mode of delivery (i.e., normal spontaneous delivery, cesarean). In the present study, adverse birth outcomes in newborns were based on LBW and low Apgar scores. LBW newborns were defined as newborns with a birth weight <2,500 g. Newborns with low Apgar scores had a 1- or 5-min Apgar score <7 [4]. Information on demographic or socioeconomic factors, or other factors that confounded the associations between maternal arsenic exposure, maternal DNA damage, and newborn birth outcomes was collected. We used an administrative questionnaire to obtain the demographic data of the pregnant women, such as age, parity, education, and health-related data (medical history, as well as cigarette smoking and alcohol use before and after pregnancy).

Statistical analysis

We assessed the associations between maternal arsenic exposure (iAs, MMA, and DMA), maternal DNA damage (8-oxodG and N7-MeG), and newborn health parameters (birth weight, birth length, head circumference, chest girth, and Apgar scores). Arsenic metabolites and DNA damage biomarker levels under LOD were recorded as one-half of the LOD values. All variables were assessed for normal distribution or natural logarithmic-transformation to approximate a normal distribution. Specifically, all urinary arsenic species levels were log-transformed, and the urinary levels of 8-oxodG and N7-MeG were natural log-transformed to obtain a normal distribution. Covariates assessed included maternal age at delivery, mode of delivery, pre-pregnancy BMI, gestational age, newborn sex, prenatal alcohol consumption, and maternal education.

To assess differences in the characteristics between subjects who were followed and subjects lost to follow-up, we used an independent sample t-test for continuous variables and the χ2 method for categorical variables (see Supplemental Material, Table S1). Pearson correlations were used to explore the associations between maternal arsenic levels, DNA damage, newborn health status, and other covariates. Significantly correlated variables were further analyzed in multivariable regression models adjusting for potential confounders. Potential confounders were identified based on correlations (p<0.1) with arsenic exposure and birth outcomes. There were no significant differences in the relationships in the birth outcomes and maternal iAs between the sexes; we combined data from female and male newborns to increase the statistical power.

Maternal age, pre-pregnant BMI, gestational age, mode of delivery, and newborn sex were included in the final model. We used birth outcomes as both continuous and binary variables in the linear regression model and the Cox's proportional hazards model, respectively. Statistical significance was set at a p<0.05. For the Cox's proportional hazards model estimates, we transformed continuous variables into a two-level scale to compare the above and below medians of the arsenic metabolites and DNA damage biomarkers. We calculated the 95% CIs of the relative risks from the corresponding regression coefficients and standard errors. All statistical analyses were performed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA).


General characteristics of the subjects

Table 1 presents the characteristics of pregnant women and the newborns by sex. Of the 299 newborns, 151 (51%) were male and 148 (49%) were female. Female newborns had significantly lower birth weights than the male newborns (3170 vs. 2985 gm, p = 0.001). Twenty-one (7%) newborns had a LBW (<2,500 g), 13 (61.9%) of which were female. Sixty-nine (23%) newborns had 1-min Apgar scores <7, of which 40 were male (p = 0.15 for sex difference). In addition, there were 4 female newborns with low 5-min Apgar scores as compared with none in males. Only 5 (1.7%) of women consumed alcohol during pregnancy. The characteristics for the excluded twins were similar to the included twins.

Table 1. Characteristics of mothers and their newborns by newborn sex in central Taiwan during 2000–2001 (n = 299).

Women lost to follow-up had a significantly lower average gestational age and greater income than the women who were followed. In addition, the mothers followed tended to be older and had slightly lower pre-pregnant BMIs than the mothers lost to follow-up (p<0.1). No significant differences existed in parity, newborn birth outcomes, arsenic levels, mode of delivery, or maternal education and life styles between the groups (see Supplemental Material Table S1).

Maternal arsenic exposure and DNA damage

The levels and distribution of arsenic metabolites in maternal urine are shown in Table 2. The median (5th–95th) levels of creatinine-adjusted urinary As3+, As5+, MMA, DMA, iAS, and tAS were 0.30 (0.06–1.77), 0.21 (0.05–2.95), 0.46 (0.07–5.82), 20.01 (1.43–69.94), 0.79 (0.18–3.96), and 22.26 (2.80–83.0) µg g cre−1, respectively. Among all arsenic metabolites, the frequency of samples with arsenic metabolite levels above the LODs was >92%. The median (5th–95th) concentrations of creatinine-adjusted urinary 8-oxodG and N7-MeG were 3.48 (1.59–8.56) µg g−1 cre and 12.88 (6.47–25.57) mg g−1, respectively (Table 2).

Table 2. Distribution of creatinine-adjusted concentrations of urinary arsenic species (iAs, MMA, and DMA) and urinary DNA damage biomarkers (8-oxodG and N7-MeG) for pregnant women in the present study versus two others with established arsenic-related effects (n = 299).

Maternal arsenic exposure, oxidative/methylated DNA damage, and birth outcomes

Table 3 shows a significant positive correlation between maternal 8-oxodG levels and iAs (r = 0.24, p<0.001), MMA (r = 0.16, p<0.001), DMA (r = 0.13, p<0.05), and tAs (r = 0.17, p<0.001). Similarly, a significant positive correlation was shown between the maternal N7-MeG levels and iAs (r = 0.35, p<0.001) and the metabolites. In addition, a significant positive correlation was shown between maternal 8-oxodG levels and N7-MeG (r = 0.62, p<0.001). A negative correlation of −0.13 (p = 0.08) was found between maternal iAs levels and the 1-min Apgar scores of newborns. A significant positive correlation was noted between the maternal MMA levels and the 1-min Apgar scores of newborns (r = 0.15, p = 0.03). A suggested negative correlation might exist between the maternal N7-MeG levels and the 1-min Apgar scores of newborns (r = −0.18, p = 0.06). The maternal MMA and DMA levels were not significantly correlated with the birth outcomes of birth weight, length, and head and chest circumferences.

Table 3. Pearson correlations between maternal inorganic arsenic and its metabolites levels, DNA damage in pregnant women, newborn health status, and other factors.

Regression analyses identified positive associations between the maternal urinary level of iAs, MMA tAs, and the DNA damage biomarkers 8-oxodG and N7-MeG (Table 4). Maternal iAs levels had negative associations with 1-min Apgar scores (β-coefficient  = −0.23, 95% CI: −0.29 − −0.18, p = 0.041). MMA had positive associations with the newborn birth weight (β = 0.25, 95% CI: 0.02–0.42, p = 0.034), and a marginally significant positive association with 1-min Apgar scores (β = 0.21, 95% CI: −0.01–0.44, p = 0.06). In addition, the DMA and tAs were not significantly associated with the 1-min Apgar score. The maternal urinary N7-MeG levels had negative associations with the birth length (β = −0.21, 95% CI: −0.86 − −0.13, p = 0.024) and 1-min Apgar score (β = −0.25, 95% CI: −0.63 − −0.15, p = 0.042). To determine whether or not the estimated effects of maternal iAs exposure on newborn health status was via methylated DNA damage, we performed a multivariable adjusted regression analysis to estimate the association between maternal iAs exposure and newborn health outcomes, controlling for N7-MeG and potential confounders (Table 5). The associations between maternal iAs levels and 1-min Apgar scores remained significant after adjusting for N7-MeG.

Table 4. Multiple linear regression (β-coefficient) for the associationa between birth outcomes with maternal urinary arsenic metabolitesb and DNA damagec biomarkers (n = 299).

Table 5. Multiple linear associations (β-coefficient) of birth outcomes with maternal urinary inorganic concentrations and DNA damage biomarkers after further mutual adjustment for each other.

Cox's proportional hazard model demonstrated that the maternal iAs level over the median was associated with the risk of decreased Apgar scores (RR = 1.14, 95% CI: 1.03–2.32, p = 0.012; Table 6). The relative risks for decreased Apgar scores was significantly increased (OR = 1.88, 95% CI: 1.07–2.15, p = 0.03) for pregnant women with urinary N7-MeG levels over the median. Relative risks for decreased Apgar scores associated with maternal N7-MeG levels were 1.92 (95% CI: 0.98–2.01, p = 0.07) after adjusting for iAs and other potential confounders.

Table 6. Relative risks (RR)a and 95% confidence interval (95% CI) for low birth weight and decreased one-minute Apgar score in relation to maternal urinary arsenic and DNA damage biomarker levels (n = 299).


This is the first study to simultaneously assess the effects from maternal arsenic exposure and maternal oxidative and methylated DNA damage on the health status of newborns. The concentrations of urinary tAs in this study were significantly lower than previous studies conducted in Bangladesh (median urinary tAs, 717.5 µg g−1 cre) [26] and Chile (median urinary tAs, 40.4 µg g−1 cre) [27]. Adverse birth outcomes and decreased 1-min Apgar scores were associated with increased maternal levels of iAs and N7-MeG in the general population.

We observed a significantly increased relative risk of low Apgar scores associated with maternal urinary iAs levels after adjusting for N7-MeG. The critical mechanism underlying prenatal arsenic exposure is still not clear. Disrupted placentation [13] and endocrine disturbance [14] have been reported for arsenic-related adverse pregnancy outcomes. Animals treated with high doses of arsenic have reported severe early effects, such as neural tube effects [28]; however, the levels of arsenic exposure relevant to humans await further investigation. DNA damage was reported to be induced by iAs, as shown by increased concentrations of the biomarkers of oxidized DNA adducts (8-oxodG) in the brains of mice [29] and in the urine of women in the early stages of pregnancy [17]. In the present study, urinary 8-oxodG andN7-MeG levels were significantly associated with concentrations of maternal urinary arsenic species. N7-MeG was significantly associated with decreased birth length and 1-min Apgar scores after adjustment for maternal age, pre-pregnant BMI, mode of delivery, gestational age, and newborn sex. Thus, N7-MeG appeared to be a more sensitive biomarker than 8-oxodG for maternal DNA damage related to newborn adverse outcomes. This suggests that maternal iAs might cause both DNA damage and adverse newborn health independent of the DNA damage.

We first reported that the association between iAs and N7-MeG. N7-MeG has been established as a marker of human exposure to methylating agents and smoking [30]. The induction of N7-MeG is believed to occur via the direct toxicity of arsenic on enzyme activities, such as DNA repair enzymes. It has been shown that low concentrations of iAs are able to inhibit the expression and activity of poly(ADP-ribose) polymerase (PARP) enzymes [31], the enzymes responsible for base excision repair pathways, which increase unrepaired DNA lesions [32]. Urinary N7-MeG is derived from transfer RNA (tRNA) turnover [33]. The current association between arsenic exposure and increased urinary N7-MeG levels might be due to upregulation of alternative repair pathways of methylated DNA damage, such as base excision repair, resulting in the formation of N7-MeG. A previous report indicated that a variety of methylated purines and pyrimidines are found in tRNA, thus degradation of tRNA might result in the release of all minor methylated bases [34]. A recent in vivo study showed that arsenic exposure induces tRNA modification in Saccharomyces cerevisiae, and may subsequently increase cellular levels of N7-MeG [35]. An improper alteration in tRNA modification may lead to disorders in embryonic development, cell proliferation, and differentiation in mice [36], and developmental abnormalities in Caenorhabditis elegans [37].

DNA methylation is an important mechanism of fetal programming during fetal development [2], [10]. Long-term exposure to arsenic in utero has been associated with changes in DNA methylation, which may have severe consequences for the development of fetal health effects [10]. Urinary N7-MeG could be used as a biomarker for the alteration of DNA methylation due to arsenic [38]. It has been shown that high arsenic exposure in pregnant mice significantly reduces DNA methylation in offspring [39]. We suggest that iAs might induce adverse birth outcomes. Further evaluation is needed in a larger sample to understand whether or not prenatal low iAs exposure results in altering DNA methylation in newborns.

Maternal urinary DMA was not significantly associated with maternal 8-oxodG nor were 1-min Apgar scores. This might result from organic arsenic as a source of DMA and tAs: A large population study showed that seafood intake was a major determinant of increased urine concentrations of DMA and total arsenic [40]. Current study area residents in Taiwan regularly consume seafood and fish from the surrounded Pacific Ocean [41]. These foods might contain substantial amounts of arsenosugars or arsenolipids, which could metabolized as DMA [41].

The main strengths of this study include a study design that allows findings on the longitudinal capture of biomarkers for inorganic arsenic exposure to relate to birth outcomes, monitoring biomarkers of oxidative damage, and sufficient sample size. In addition, none of the study subjects smoked cigarettes, which otherwise would have been a major confounder because smoking is associated with several adverse outcomes of pregnancy, including LBW [42] and increased N7-MeG levels [30].

The current study was limited by the lack of paternal data, thus early contributions from the male complement due to arsenic-induced damage cannot be evaluated and warrant further studies. Furthermore, iAs has a short half-life and the efficiency of inorganic arsenic methylation to DMA was increasing since the 1st trimester of pregnancy [43]. We only measured urinary arsenic species once during the third trimester, which likely introduced a substantial degree of exposure measurement misclassification towards null hypothesis.

In this study, there were no significant differences in the association of birth outcomes with maternal iAs between sexes; we combined data from female and male newborns to increase the statistical power. Future work might recruit more subjects to further verify the sex difference. We assessed arsenic and biomarkers of DNA damage using the same spot urine and thus temporality cannot be evaluated. However, we do not think the converse is likely to occur (i.e., greater oxidation/methylation metabolism increases arsenic elimination).


This is the first study to report significant associations between arsenic-induced N7-MeG levels of pregnant women and an increased risk of adverse birth outcomes in newborns. These findings strongly emphasize that maternal N7-MeG may be a sensitive and effective biomarker for newborn health, particularly after early-life arsenic exposure. Further studies are necessary to understand the potential health effects of arsenic-related DNA methylation or tRNA modifications in newborns.

Supporting Information

Table S1.

Demographic characteristic of followed and lost-to-follow-up subjects.




We are greatly indebted to the clinics of the Department of Pediatrics and Department of Obstetrics and Gynecology, Chung-Shan Medical University Hospital, especially for the assistance of Dr. Jia-Yuh Chen, Dr. Long-Yau Lin, Dr. Pen-Hua Su, and Dr. Chin-Yi Lin. We appreciate the earlier participation of Mr. Yun-Siang Liu and Dr. Po-Chin Huang at the National Health Research Institutes, Miaoli, Taiwan, including subject recruitment and specimen processing.

Author Contributions

Conceived and designed the experiments: SLW YCT. Performed the experiments: WCC YTC CJW YCT. Analyzed the data: WCC. Contributed reagents/materials/analysis tools: WCC YTC HYC CJW YCT. Wrote the paper: WCC SLW. Providing clinical materials: THY. Advising the 1st author: CYC SLW.


  1. 1. Doctor BA, O'Riordan MA, Kirchner HL, Shah D, Hack M (2001) Perinatal correlates and neonatal outcomes of small for gestational age infants born at term gestation. Am J Obstet Gynecol 185: 652–659.
  2. 2. Valdez R, Athens MA, Thompson GH, Bradshaw BS, Stern MP (1994) Birth weight and adult health outcomes in a biethnic population in the USA. Diabetologia 37: 624–631.
  3. 3. Kajantie E, Osmond C, Barker DJP, Forsén T, Phillips DIW, et al. (2005) Size at birth as a predictor of mortality in adulthood: a follow-up of 350 000 person-years. Int J Epidemiol 34: 655–663.
  4. 4. Casey BM, McIntire DD, Leveno KJ (2001) The continuing value of the Apgar score for the assessment of newborn infants. New Engl J Med 344: 467–471.
  5. 5. Saenger P, Czernichow P, Hughes I, Reiter EO (2007) Small for gestational age: short stature and beyond. Endocr Rev 28: 219–251.
  6. 6. Odd DE, Rasmussen F, Gunnell D, Lewis G, Whitelaw A (2007) A cohort study of low Apgar scores and cognitive outcomes. Arch Dis Child Fetal Neonatal Ed 93: F115–F120.
  7. 7. Concha G, Vogler G, Lezcano D, Nermell B, Vahter M (1998) Exposure to inorganic arsenic metabolites during early human development. Toxicol Sci 44: 185–190.
  8. 8. Rahman A, Vahter M, Smith AH, Nermell B, Yunus M, et al. (2009) Arsenic exposure during pregnancy and size at birth: a prospective cohort study in Bangladesh. Am J Epidemiol 169: 304–312.
  9. 9. Huyck KL, Kile ML, Mahiuddin G, Quamruzzaman Q, Rahman M, et al. (2007) Maternal arsenic exposure associated with low birth weight in Bangladesh. J Occup Environ Med 49: 1097–1104.
  10. 10. Yang CY, Chuang HY, Ho CK, Wu TN, Wu MTF (2003) Arsenic in drinking water and adverse pregnancy outcome in an arseniasis-endemic area in Northeastern Taiwan. Epidemiology 14: S127–S127.
  11. 11. Myers SL, Lobdell DT, Liu Z, Xia Y, Ren H, et al. (2010) Maternal drinking water arsenic exposure and perinatal outcomes in inner Mongolia, China. J Epidemiol Community Health 64: 325–329.
  12. 12. Vahter M (2009) Effects of arsenic on maternal and fetal health. Annu Rev Nutr 29: 381–399.
  13. 13. He WJ, Greenwell RJ, Brooks DM, Calderon-Garciduenas L, Beall HD, et al. (2007) Arsenic exposure in pregnant mice disrupts placental vasculogenesis and causes spontaneous abortion. Toxicol Sci 99: 244–253.
  14. 14. Davey JC, Nomikos AP, Wungjiranirun M, Sherman JR, Ingram L, et al. (2008) Arsenic as an endocrine disruptor: Arsenic disrupts retinoic acid receptor- and thyroid hormone receptor-mediated gene regulation and thyroid hormone-mediated amphibian tail metamorphosis. Environ Health Perspect 116: 165–172.
  15. 15. Vijayaraghavan M, Wanibuchi H, Karim R, Yamamoto S, Masuda C, et al. (2001) Dimethylarsinic acid induces 8-hydroxy-2′-deoxyguanosine formation in the kidney of NCI-Black-Reiter rats. Cancer Lett 165: 11–17.
  16. 16. Yamanaka K, Mizol M, Kato K, Hasegawa A, Nakano M, et al. (2001) Oral administration of dimethylarsinic acid, a main metabolite of inorganic arsenic, in mice promotes skin tumorigenesis initiated by dimethylbenz(a)anthracene with or without ultraviolet B as a promoter. Biol Pharm Bull 24: 510–514.
  17. 17. Engstrom KS, Vahter M, Johansson G, Lindh CH, Teichert F, et al. (2010) Chronic exposure to cadmium and arsenic strongly influences concentrations of 8-oxo-7,8-dihydro-2′-deoxyguanosine in urine. Free Radic Biol Med 48: 1211–1217.
  18. 18. Ahmed S, Mahabbat-e Khoda S, Rekha RS, Gardner RM, Ameer SS, et al. (2011) Arsenic-associated oxidative stress, inflammation, and immune disruption in human placenta and cord blood. Environ Health Perspect 119: 258–264.
  19. 19. Chiang SY, Huang TH, Uang SN, Wu HDI, Wei YC, et al. (2005) Analysis of 7-methylguanine using isotope dilution and gas chromatography/electron-capture negative chemical ionization mass spectrometry. Rapid Commun Mass Spectrom 19: 1915–1920.
  20. 20. Rydberg B, Lindahl T (1982) Nonenzymatic methylation of DNA by the intracellular methyl group donor S-adenosyl-L-methionine is a potentially mutagenic reaction. EMBO J 1: 211–216.
  21. 21. Wang SL, Lin CY, Guo YLL, Lin LY, Chou WL, et al. (2004) Infant exposure to polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls (PCDD/Fs, PCBs)—correlation between prenatal and postnatal exposure. Chemosphere 54: 1459–1473.
  22. 22. Chou WC, Chuang CY, Huang PC, Wang CJ, Chen HY, Chuang YD, et al.. (2012) Arsenic exposure in pregnancy increases the risk of adverse birth outcomes of newborn in Taiwan. In: Ng JC, Noller BN, Naidu R, Bundschuh J, editors. Proceedings of the 4th International Congress: Arsenic in the Environment, 2012.Cairns, Australia. pp. 157–158.
  23. 23. Ritsema R, Dukan L, Navarro TRI, van Leeuwen W, Oliveira N, et al. (1998) Speciation of arsenic compounds in urine by LC-ICP MS. Appl Organometal Chem 12: 591–599.
  24. 24. Chao MR, Wang CJ, Wu MT, Pan CH, Kuo CY, et al. (2008) Repeated measurements of urinary methylated/oxidative DNA lesions, acute toxicity, and mutagenicity in coke oven workers. Cancer Epidemiol Biomarkers Prev 17: 3381–3389.
  25. 25. Baskett TF (2000) Virginia Apgar and the newborn Apgar score. Resuscitation 47: 215–217.
  26. 26. Hall M, Gamble M, Slavkovich V, Liu X, Levy D, et al. (2007) Determinants of arsenic metabolism: blood arsenic metabolites, plasma folate, cobalamin, and homocysteine concentrations in maternal-newborn pairs. Environ Health Perspect 115: 1503–1509.
  27. 27. Christian WJ, Hopenhayn C, Centeno JA, Todorov T (2006) Distribution of urinary selenium and arsenic among pregnant women exposed to arsenic in drinking water. Environ Res 100: 115–122.
  28. 28. Hill DS, Wlodarczyk BJ, Finnell RH (2008) Reproductive consequences of oral arsenate exposure during pregnancy in a mouse model. Birth Defects Res B Dev Reprod Toxicol 83: 40–47.
  29. 29. Piao F, Ma N, Hiraku Y, Murata M, Oikawa S, et al. (2005) Oxidative DNA damage in relation to neurotoxicity in the brain of mice exposed to arsenic at environmentally relevant levels. J Occup Health 47: 445–449.
  30. 30. Lin IH, Chao MR, Hu CW, Ho ML, Huang JY, et al. (2009) Modification of urinary N7-methylguanine excretion in smokers by glutathione-S-transferase M1 polymorphism. Toxicology 260: 1–6.
  31. 31. Walter I, Schwerdtle T, Thuy C, Parsons JL, Dianov GL, et al. (2007) Impact of arsenite and its methylated metabolites on PARP-1 activity, PARP-1 gene expression and poly(ADP-ribosyl)ation in cultured human cells. DNA Repair (Amst) 6: 61–70.
  32. 32. Qin XJ, Liu W, Li YN, Sun X, Hai CX, et al. (2012) Poly(ADP-ribose) polymerase-1 inhibition by arsenite promotes the survival of cells with unrepaired DNA lesions induced by UV Exposure. Toxicol Sci 127: 120–129.
  33. 33. Shuker DEG, Farmer PB (1992) Relevance of urinary DNA adducts as markers of carcinogen exposure. Chem Res Toxicol 5: 450–460.
  34. 34. Mandel LR, Srinivasan PR, Borek E (1966) Origin of urinary methylated purines. Nature 209: 586–588.
  35. 35. Chan CTY, Dyavaiah M, DeMott MS, Taghizadeh K, Dedon PC, et al. (2010) A quantitative systems approach reveals dynamic control of tRNA modifications during cellular stress. PLoS Genet 6: e1001247.
  36. 36. Tuorto F, Liebers R, Musch T, Schaefer M, Hofmann S, et al. (2012) RNA cytosine methylation by Dnmt2 and NSun2 promotes tRNA stability and protein synthesis. Nat Struct Mol Biol 19: 900–905.
  37. 37. Chen CC, Tuck S, Bystrom AS (2009) Defects in tRNA modification associated with neurological and developmental dysfunctions in Caenorhabditis elegans elongator mutants. PLoS Genet 5: e1000561.
  38. 38. Stillwell WG, Xu HX, Adkins JA, Wishnok JS, Tannenbaum SR (1989) Analysis of methylated and oxidized purines in urine by capillary gas-chromatography mass-spectrometry. Chem Res Toxicol 2: 94–99.
  39. 39. Xie YX, Liu J, Benbrahim-Tallaa L, Ward JM, Logsdon D, et al. (2007) Aberrant DNA methylation and gene expression in livers of newborn mice transplacentally exposed to a hepatocarcinogenic dose of inorganic arsenic. Toxicology 236: 7–15.
  40. 40. Navas-Acien A, Francesconi KA, Silbergeld EK, Guallar E (2011) Seafood intake and urine concentrations of total arsenic, dimethylarsinate and arsenobetaine in the US population. Environ Res 111: 110–8.
  41. 41. Lee MM, Pan WH, Yu SL, Huang PC (1992) Foods predictive of nutrient intake in Chinese diet in Taiwan: I. Total calories, protein, fat and fatty acids. Int J Epidemiol 21: 922–928.
  42. 42. Bjerg A, Hedman L, Perzanowski M, Lundbäck B, Rönmark E (2011) A strong synergism of low birth weight and prenatal smoking on asthma in schoolchildren. Pediatrics 127: E905–E912.
  43. 43. Gardner RM, Nermell B, Kippler M, Grandér M, Li L, Ekström EC, et al. (2011) Arsenic methylation efficiency increases during the first trimester of pregnancy independent of folate status. Reprod Toxicol 31: 210–218.