Environmental Pollution 263 (2020) 114607

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Environmental Pollution

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Cross-sectional and longitudinal associations between global DNA (hydroxy) methylation and exposure biomarkers of the Hebei Spirit oil spill cohort in Taean, Korea*

Nivedita Chatterjee a, Jaeseong Jeong a, Myung-Sook Park b, Mina Ha c, * Hae-Kwan Cheong d, Jinhee Choi a, a School of Environmental Engineering, University of Seoul, 163 Seoulsiripdae-ro, Dongdaemun-gu, Seoul, 02504, South Korea b Taean Environmental Health Center, Taean, Chungnam, 32148, South Korea c Department of Preventive Medicine, Dankook University College of Medicine, Cheonan, Chungnam, 31116, South Korea d Department of Social and Preventive Medicine, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi, 16419, South Korea article info abstract

Article history: The Hebei Spirit oil spill (HSOS) occurred on the west coast of South Korea (Taean county) on December Received 8 October 2019 7, 2007, and studies revealed that exposure to the oil spill was associated with various adverse health Received in revised form issues in the inhabiting population. However, no studies evaluated the association between crude-oil 25 March 2020 exposure and epigenetic changes. This study aimed to investigate the HSOS exposure-associated longi- Accepted 14 April 2020 tudinal and cross-sectional variations in global DNA methylation (5-mc) and/or hydroxymethylation (5- Available online 18 April 2020 hmc) and expression profiles of related genes in Taean cohort participants from 2009 (AH-baseline) and 2014 (AH-follow-up) relative to the reference group (AL). We measured global DNA 5-mc and 5-hmc Keywords: fi fi Hebei Spirit oil spill adult cohort levels and related gene expression levels in whole blood. We identi ed signi cant associations be- DNA methylation (5-mc) tween HSOS exposure and AH-baseline-5-mc, AH-baseline-5-hmc, and AH-follow-up-5-hmc. HSOS DNA hydroxymethylation (5-hmc) exposure was associated with lower %5-mc content and higher %5-hmc content in the same individuals DNMT3B from both the cross-sectional and longitudinal studies. In addition, we found a strong correlation be- TET1 tween 5-mc and DNMT3B expression, and between 5-hmc and TET1 expression. Our findings suggested that epigenetic changes are important biomarkers for HSOS exposure and that 5-hmc is likely to be more sensitive for environmental epidemiological studies. © 2020 Elsevier Ltd. All rights reserved.

1. Introduction City. Shortly after the accident, many volunteers, most of which were local residents with military personnel, participated in longer The Hebei Spirit oil spill (HSOS) occurred on the west coast of cleanup activities for periods ranging from days to several months South Korea (Taean county) on December 7, 2007 and was (Ha et al., 2012). During the early clean-up work, some of the par- considered not only one of the worst oil spills in Korea, but also one ticipants lacked protective equipment and were exposed to oil- of the most serious oil spills in the history of East Asia (Yim et al., related chemicals. Previous studies on the Taean cohort suggested 2012). The spill size was relatively small (estimated 10,900 tons adverse health effects, such as the increased levels of oxidative of crude oil) compared to those of other major oil spill accidents stress biomarkers (MDA and 8-OHdG) and PAH metabolites (1-OHP worldwide (e.g., the Exxon Valdez spilled 37,000 tons of crude oil) and 2-NAPH) in clean-up participants, which were found to be (Kim et al., 2017). However, the main concern regarding the HSOS correlated with the duration of clean-up work in a cross-sectional was that the accident occurred near a heavily populated area, only study (Eom et al., 2011; Ha et al., 2012; Kim et al., 2017; Noh about 10 km away from the beach and the residential area of Taean et al., 2015). In general, crude oil contains various substances, including volatile organic compounds (VOCs), polycyclic aromatic hydrocar- bons (PAHs), and heavy metals. The majority of the components * This paper has been recommended for acceptance by Christian Sonne. present in crude oil are classified under group 1 (carcinogenic), * Corresponding author. E-mail address: [email protected] (J. Choi). group 2A (probably carcinogenic), and group 2B (possibly https://doi.org/10.1016/j.envpol.2020.114607 0269-7491/© 2020 Elsevier Ltd. All rights reserved. 2 N. Chatterjee et al. / Environmental Pollution 263 (2020) 114607 carcinogenic) chemicals in International Agency for Research on participate clean-up work for AL group. Finally, 30 persons for AL Cancer (IARC) monograms (Choi et al., 2018; Park et al., 2017; Yang (AL-follow-up group) and 32 for AH (AH-baseline as well as AH- et al., 2017). follow-up groups) were included in the present study. The study The ‘induction of epigenetic alterations’ is one of ten key char- protocol was approved by the Institutional Review Board of Dan- acteristics of carcinogens described in IARC monographs 112 and kook University Hospital, and informed consent was obtained from 113 (Smith et al., 2016). An increasing number of studies reported a all subjects (IRB no. 0904-027, 2014-06-013). significant association between epigenetic biomarkers and disease. Alterations in epigenetic biomarkers, particularly global DNA 2.2. Questionnaire methylation levels, including 5-methylcytosine (5-mc) and 5- hydroxymethylcytosine (5-hmc) levels, and the expression levels Demographic information was obtained through self-reported of their regulatory genes are associated the incidence of various questionnaire each surveys. Socio-demographic factors, i.e., age at types of cancer (Fouad et al., 2018; Joyce et al., 2016; Li et al., 2017; the oil spill accident (<60, 60), gender (male, female), healthy Lian et al., 2012; Sarabi and Naghibalhossaini, 2015; Tucker et al., behavior, i.e., smoking status (never smoker, current smoker, ex- 2018; Wilson et al., 2007). smoker) were considered. Participants were asked about duration On the other hand, the epigenetic alterations showed a relation of participated cleanup-works. The distance from the oil spill site with various environmental and occupational exposure (Cardenas was calculated using the ArcGIS Desktop ver.9.3 geographic infor- et al., 2017, 2015; Martin and Fry, 2018; Sanchez-Guerra et al., mation system (ESRI, Redlands, CA, USA), based on the latitude and 2015; Tellez-Plaza et al., 2014) and the incidence of various types longitude of the residential address, and considered when selecting of cancer (Fouad et al., 2018; Joyce et al., 2016; Li et al., 2017; Lian the group. et al., 2012; Sarabi and Naghibalhossaini, 2015; Tucker et al., 2018; Wilson et al., 2007). Nonetheless, the association of 5-mc 2.3. Global DNA methylation (5-mc) and global DNA and 5-hmc levels with specific biological changes caused by envi- hydroxymethylation (5-hmc) measurements ronmental exposure has not been well explored in epidemiological studies, except for a few recent studies (Cardenas et al., 2017; Total DNA from whole blood samples were extracted using a Sanchez-Guerra et al., 2015; Tellez-Plaza et al., 2014). DNA extraction kit (NucleoSpin, Macherey-Nagel). DNA quality and No studies have investigated epigenetic changes, such as global quantity were evaluated using a micro-volume spectrophotometer DNA methylation levels following crude oil spill exposure, and their (ASP-2680, ACTGene, Piscataway, NJ, USA). Next, global DNA associations with biomarkers for exposure or health effects. methylation [MethylFlash Global DNA Methylation (5-mc) ELISA In the present study, we analyzed the associations and corre- Easy Kit (Colorimetric)] and global DNA hydroxymethylation lations among global DNA methylation (5-mc) and DNA hydrox- [MethylFlash Global DNA Hydroxymethylation (5-hmc) ELISA Easy ymethylation (5-hmc) markers, expression levels of associated Kit (Colorimetric)] assays were carried out according to the man- genes (DNMTs, TETs, and MBDs), oxidative stress biomarkers (8- ufacturer’s instructions using an ELISA kit (Epigentek Group Inc., OHdG and MDA), and DNA repair genes (OGG1, MHT1, ERCC1, NY, USA). Briefly, 1e8 mL (100 ng) of DNA was added to the wells. XPA, and XPC) in subsamples of longitudinal studies (sampling and The samples were incubated at 37 C for 90 min, followed by the assessment for two time points) of the HSOS in Taean, Korea. addition of capture and detection antibodies. The absorbance was read at 450 nm. A standard curve was generated from the plot of OD 2. Materials and methods values relative to those of the positive controls.

2.1. Study population and sample (urine and blood) collection 2.4. Measurement of MDA and 8-OHdG levels

The number of adults (aged 18 years or older) who agreed to We used the existing results of MDA and 8-OHdG levels participate in the survey of the cohort study were 9585 from 2009 measured in the previous study of the cohort (Noh et al., 2015). to 2017. From the high exposed area (AH), the number of partici- Briefly, urinary MDA levels were determined by measuring the pants in 2009 (the baseline survey) was 1741 whereas in 2014 (the levels of thiobarbituric acid reactive substances using a high- follow-up survey) only 615 adults were participated (Fig. 1). The performance liquid chromatography instrument equipped with a references of this cohort study was from the low exposed area of fluorescence detector (Agarwal and Chase, 2002). Urinary 8-OHdG same county with that of high exposed area but distant from the levels were determined using an 8-OHdG competitive enzyme- oil-spill accident site. The adult participants (AL) from low-exposed linked immunosorbent assay (ELISA) kit (Japan Institute for the group were 7505 at baseline survey in 2009 while only 187 adults Control of Aging, Kyoto, Japan) (Noh et al., 2015). were participated in follow-up survey in 2014 (Fig. 1). The participants were administered questionnaire and under- 2.5. Total RNA extraction and quantitative real-time PCR (qRT-PCR) taken health examination with biospecimen collection at each survey. Participants were instructed to collect urine samples after a Total RNA whole blood samples were extracted using an RNA 12 h fasting period between dinner and breakfast of the following extraction kit (NucleoSpin, Macherey-Nagel). RNA quantity and morning. Samples were immediately stored in a freezer at 20 C quality were assessed using a micro-volume spectrophotometer until analysis. Blood samples were obtained from participants after (ASP-2680, ACTGene, Piscataway, NJ, USA) and by conducting a 12 h fast and stored in a freezer at 80 C. The details of the cohort agarose gel electrophoresis. cDNA synthesis was performed by study protocol was described elsewhere (Park et al., 2019). running a reverse transcriptase (RT) reaction, and PCR amplifica- In the present study, we selected subpopulation from AL and AH, tion was carried out on a thermal cycler (Bio-Rad). Real-time PCR considering completeness of questionnaire response, availability of (RT-PCR) analysis was performed using a CFX manager (Bio-Rad) exposure biomarkers measured, sufficiency of collected bio- and the IQTM SYBR Green SuperMix (Bio-Rad). The primers were specimen. We also applied additional inclusion criteria for each designed using Primer3plus (Table S1) based on the sequences group; the persons who participated in clean-up work for more available in NCBI. qRT-PCR conditions were optimized (efficiency than 200 days and were repeatedly surveyed both in the baseline and sensitivity tests) for each primer pair prior to the experiment. and follow-up for AH group, and the persons who did not All samples were measured in triplicate, and the average values N. Chatterjee et al. / Environmental Pollution 263 (2020) 114607 3

Fig. 1. Study area, study population and sampling flowchart of Hebei Spirit oil spill exposed adult and reference adult group of Taean cohort, Korea. were reported. Analysis of negative control reactions (minus RT and et al., 2018); and v) corrplot (Wei and Simko, 2017). all reagents minus template) confirmed the absence of DNA contamination. Gene expression levels were normalized against GAPDH and ACTB expression levels. 3. Results

Epidemiological characteristics, such as age, gender, smoking 2.6. Statistical analysis status, of the HSOS cohort are presented in Table 1. The mean (±SD) age of the study sample was 58.3 ± 9.1 years, out of which 43.8% The significance of differences of characteristics between groups were women. As the significant difference was observed in gender or time was determined by using a nonparametric Mann-Whitney between AL (referent) and AH (HSOS-exposed) groups, adjustment test and Wilcoxon-rank sum test. To consider the difference of analysis was performed. Gender adjusted values of level of global gender distribution between groups, we estimated the adjusted DNA methylation (%5-mc and %5-hmc) and related gene expres- values by calculation of least square mean based on the generalized sions in referents and exposed subjects from HSOS cohort is pre- linear model (GLM) including gender status as an independent sented in Table 2 (Unadjusted data are presented in Table S2 and the variable after the log transformation of the data and verifying the results were not materially different). normality using the Shapiro-Wilk test. Paired Wilcoxon-rank sum Compared to those of the AL group, we observed significantly comparison analysis was conducted between baseline and follow- reduced global DNA methylation (%5-mc) levels in AH groups with up in AH group. Spearman correlation coefficients (rs) were calcu- an additional reduction over time (baseline to follow-up) (Table 2 lated to determine the correlation among all measured parameters and Fig. S1). On the other hand, the level of global DNA hydrox- between groups: i) global DNA methylation (%5-mc) and global ymethylation (%5-hmc) was not significantly different between AL DNA hydroxymethylation (%5-hmc) levels; ii) global DNA methyl- and AH-baseline, however, which showed a significant increase in ation (%5-mc) and the DNMT3B gene expression levels; and iii) AH-follow-up (Table 2 and Fig. S1). The ratio between 5-mc and 5- global DNA hydroxymethylation (%5-hmc) levels and TET1 hmc levels was markedly lower compared to that of the AH- expression levels. Paired Spearman correlation analysis was con- baseline (93.76) to AH-follow-up (48.75) group; and both param- ducted between baseline and follow-up in AH group. Results were eters were found to be significantly lower compared to those of the presented as correlogram, histogram, scatter plots, and lowess AL group (137.40) (Table 2). models and provided as the Additional file. Linear regression The treatment groups showed significant differences in DNMT3B analysis was performed to evaluate the associations of global DNA and MBD3 expression levels. The order of DNMT3B gene expression methylation and DNA hydroxymethylation levels with the length of levels was AL > AH-baseline > AH-follow-up, while the order of participation (days) in the HSOS cleanup. Two-sided tests were MBD3 gene expression levels was AL > AH-follow-up > AH-baseline performed, and p < 0.05 was considered statistically significant. (Table 2 and Fig. S2). Distinct upregulation of DNMT1 and MBD2 The correlation analyses were performed in R software, R 3.5.1 levels was evident among the groups. However, MBD1 expression (www.r-project.org). The following R packages were used in this was significantly downregulated in the HSOS-exposed groups (AH) study: i) dplyr (Wickham et al., 2018); ii) ggplot2 (Wickham, 2009); relative to that in the reference group (AL) (Table 2 and Fig. S2). iii) Hmisc (Harrell Jr, 2018); iv) PerformanceAnalytics (Peterson Conversely, TET1 and MBD2 expression levels were markedly 4 N. Chatterjee et al. / Environmental Pollution 263 (2020) 114607

Table 1 Epidemiological characteristics with measured biomarkers in the participants of the Hebei Spirit oil spill at baseline, Taean cohort, Korea.

Epidemiological characteristics AL (n ¼ 30) a AH (n ¼ 32) b p-value d

Age (years) Overall 58.1 ± 11.8 58.3 ± 9.1 0.569 <60 12 (40) 14 (43.8) 0.765 60 18 (60) 18 (56.2) Gender Female 25 (83.3) 14 (43.8) <0.001 Male 5 (16.7) 18 (56.2) Smoking Never smoker 26 (83.3) 20 (62.5) 0.076 Current smoker 2 (6.7) 9 (28.1)c Ex-smoker 2 (6.7) 3 (9.4)c Participation in oil cleaning (days) N/A 261.4 ± 40.9

Mean ± SD or Number (%). AL; adults low exposed, AH; adults high exposed. a Referent subjects (low/no exposed group) who are residents distant from the accident site and did not take part in oil cleaning. b High exposed group who are residents close to the accident site and took part in oil cleaning for more than 200 days. c Three current smokers at baseline were changed to ex-smokers at the follow-up. d P-value estimated using Wilcoxon-test and Chi-squared test or Fisher’s exact test.

Table 2 Table 3 Cross-sectional and longitudinal difference on the levels of global DNA methylation Cross-sectional and longitudinal difference on the levels of oxidative stress bio- (%5-mc and %5-hmc) and related gene expressions adjusted for gender status in markers, and DNA repair gene expressions adjusted for gender status in referents referents and exposed subjects from the Hebei Spirit oil spill, Taean cohort, Korea. and exposed subjects from the Hebei Spirit oil spill, Taean cohort, Korea.

a b Biomarkers AL (n ¼ 30) a AH (n ¼ 32) b Biomarkers AL (n ¼ 30) AH (n ¼ 32)

Baseline, 2009 Follow-up, 2014 Baseline, 2009 Follow-up, 2014 c c c Mean ± SD c Mean ± SD c Mean ± SD c Mean ± SD Mean ± SD Mean ± SD

Global DNA methylation status Oxidative stress biomarkers yy %5-mc 5.01 ± 1.44 3.52 ± 1.26 ** 2.43 ± 1.43 ** MDA 1.63 ± 1.62 2.13 ± 2.07 * 2.43 ± 1.82 ** yy y %5-hmc 0.036 ± 1.12 0.037 ± 1.24 0.050 ± 1.29 ** 8-OHdG 3.75 ± 1.59 4.76 ± 1.75 6.17 ± 1.58 ** yy 5-mc to 5-hmc ratio 137.40 ± 1.46 93.76 ± 1.47 ** 48.75 ± 1.72 ** DNA repair gene expression (Base-excision repair) yy DNA methylation gene expression (DNA methyltransferase) OGG1 2.02 ± 1.39 5.02 ± 1.26 ** 3.33 ± 1.53 ** yy yy DNMT1 1.41 ± 1.47 2.55 ± 1.29 ** 1.57 ± 1.71 MTH1 1.81 ± 1.58 2.56 ± 1.44 * 1.56 ± 1.75 DNMT3A 2.09 ± 1.46 2.48 ± 1.26 2.08 ± 1.58 DNA repair gene expression (Nucleotide-excision-repair) yy yy DNMT3B 0.83 ± 1.49 0.53 ± 1.67 ** 0.34 ± 1.81 ** ERCC1 2.77 ± 1.33 5.90 ± 1.33 ** 3.32 ± 1.36 * yy DNA methylation gene expression (methyl-CpG binding domain protein) XPA 1.41 ± 2.00 4.92 ± 1.44 ** 2.77 ± 1.39 ** yy MBD1 6.01 ± 1.29 3.17 ± 1.43 ** 2.48 ± 1.40 ** XPC 2.31 ± 1.43 2.30 ± 1.55 2.25 ± 1.49 MBD2 2.02 ± 1.48 2.67 ± 1.51 * 3.02 ± 1.52 ** fi yy Signi cant difference between baseline and follow-up in AH group by paired ± ± ** ± ** y yy MBD3 0.96 1.41 0.30 1.51 0.41 2.11 comparison @ p-value <0.05 and <0.01 . DNA methylation gene expression (methyl-CpG binding protein 2) AL; adults low exposed, AH; adults high exposed. ± ± ± MECP2 3.18 1.33 3.39 1.34 3.62 1.37 a Referent subjects (low/no exposed group) who are residents distant from the DNA demethylation gene expression accident site and did not take part in oil cleaning. ± ± ** ± ** TET1 1.61 1.48 3.98 1.68 3.92 1.56 b High exposed group who are residents close to the accident site and took part in ± ± * ± ** TET2 1.50 1.46 2.00 1.39 2.05 1.56 oil cleaning for more than 200 days. c AL; adults low exposed, AH; adults high exposed. Mean and standard deviation were calculated by using least square mean based Significant difference between baseline and follow-up in AH group by paired on the generalized linear model adjusted for gender status, significantly different y yy comparison @ p-value <0.05 and <0.01 . between AL and AH groups @ p-value <0.05* and <0.01**. a Referent subjects (low/no exposed group) who are residents distant from the accident site and did not take part in oil cleaning. b High exposed group who are residents close to the accident site and took part in and Fig. S3). Similar to the BER genes, the selected nucleotide- oil cleaning for more than 200 days. c excision-repair (NER) genes (ERCC1 and XPA) were markedly Mean and standard deviation were calculated by using least square mean based fi on the generalized linear model adjusted for gender status, significantly different upregulated in HSOS-exposed subjects (AH), speci cally in AH- between AL and AH groups @ p-value <0.05* and <0.01**. baseline, compared to those of the reference subjects (AL), except XPC (Table 3 and Fig. S3). In general, the order of expression levels of DNA-repair genes (mean ± SD) was AH-baseline > AH-follow- upregulated in the HSOS-exposed groups (AH) compared to those up > AL groups (Table 3). in the reference group (AL). The AH-baseline and -follow-up, and Pairwise correlations between all measured biomarkers were AL groups showed no significant differences in DNMT3A and MECP2 presented as a correlogram and histogram for the AH-baseline expression levels (Table 2 and Fig. S2). (Fig. S4A and Fig. S4B) and AH-follow-up (Fig. S5A and Fig. S5B) Gender adjusted values of level of oxidative stress biomarkers groups. Global DNA %5-mc and %5-hmc levels were significantly and DNA-repair genes expressions in referents and exposed sub- correlated; the Spearman’s correlation coefficients were 0.375 jects from HSOS cohort is presented in Table 3 (Unadjusted data are (p ¼ 0.0118) and 0.574 (p ¼ 0.0018) for the AH-baseline and AH- presented in Table S3). Notably, the urinary oxidative stress follow-up groups, respectively (Fig. 2). The observed patterns for biomarker levels (8-OHdG and MDA) consistently increased over both epigenetic modifications (5-mc and 5-hmc) were consistent in time in both HSOS-exposed groups (baseline and follow-up) rela- both the cross-sectional and longitudinal studies, with few excep- tive to those of the reference groups (AL) (Table 3 and Fig. S1). tions (Fig. 2 and Fig. S1). Among the measured writer-reader- Consistent with the increased 8-OHdG levels, expression levels of erasers of global DNA methylation, we found a strong and signifi- base excision repair (BER) genes (OGG1 and MTH1) were markedly cant correlation between 5-mc levels and DNMT3B expression (AH- upregulated compared to those in the reference group (AL) (Table 3 baseline: rs ¼ 0.64, p < 0.0001; AH-follow-up: rs ¼ 0.494, N. Chatterjee et al. / Environmental Pollution 263 (2020) 114607 5

Fig. 2. Association (Spearman’s correlation coefficient, rs) of global DNA methylation (%5-mc) and global DNA hydroxymethylation (%5-hmc) in Hebei Spirit oil spill exposed adult group (n ¼ 32) in Taean cohort, Korea. AH-baseline ¼ samples collected at 2009; AH-follow-up ¼ samples collected at 2014. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3. Association (Spearman’s correlation coefficient, rs) of global DNA methylation (%5-mc) and DNMT3B gene expressions in Hebei Spirit oil spill exposed adult group (n ¼ 32) in Taean cohort, Korea. AH-baseline ¼ samples collected at 2009; AH-follow-up ¼ samples collected at 2014. *p < 0.05, **p < 0.01, ***p < 0.001. p ¼ 0.0013) (Fig. 3) and between 5-hmc levels and TET1 expression than those in the reference group (Table 1). To identify the asso- (AH-baseline: rs ¼ 0.678, p ¼ 0.0008; AH-follow-up: rs ¼ 0.522, ciation between methylation (5-mc/5-hmc) and exposure, we p < 0.0001) (Fig. 4) for both the AH-baseline and AH-follow-up conducted regression analysis considering the number of ‘cleaning groups. A significant correlation was identified between clean up days’ as independent variable. We identified a significant correla- participation and AH-baseline-5-mc (rs ¼0.453, p ¼ 0.0042), AH- tion between the number of cleaning days and AH-baseline-5-mc, baseline-5-hmc (rs ¼ 0.603, p < 0.0001), and AH-follow-up-5-hmc AH-baseline-5-hmc, and AH-follow-up-5-hmc pairs (Fig. S6). levels (rs ¼ 0.276, p ¼ 0.03) (Fig. 5). However, no significant cor- relations were found between the number of clean up days with 8- OHdG levels and between 8-OHdG levels and expression levels of 4. Discussion DNA repair genes (OGG1, MTH1, ERCC1, XPA, and XPC)(Fig. S4 and Fig. S5), even though the 8-OHdG levels were consistently higher We designed the longitudinal study (two time points, 2009 and 2014) comprising 32 subjects with high exposure to oil spill and 30

Fig. 4. Association (Spearman’s correlation coefficient, rs) of global DNA hydroxymethylation (%5-hmc) and TET1 gene expressions in Hebei Spirit oil spill exposed adult group (n ¼ 32) in Taean cohort, Korea. AH-baseline ¼ samples collected at 2009; AH-follow-up ¼ samples collected at 2014. *p < 0.05, **p < 0.01, ***p < 0.001. 6 N. Chatterjee et al. / Environmental Pollution 263 (2020) 114607

Fig. 5. Association (Spearman’s correlation coefficient, rs) of global DNA methylation (%5-mc) and global DNA hydroxymethylation (%5-hmc) with the participation in cleaning works in Hebei Spirit oil spill exposed adult group (n ¼ 32) in Taean cohort, Korea. AH-baseline ¼ samples collected at 2009; AH-follow-up ¼ samples collected at 2014. *p < 0.05, **p < 0.01, ***p < 0.001. subjects with low-exposure (referents) to evaluate whether crude 2014). In our study population, we identified a consistent associa- oil exposure cause changes in 5-mc and/or 5-hmc levels and tion between global 5-mc and 5-hmc levels, persistently high levels oxidative stress (8-OHdG), in the subjects. In addition, we of oxidative stress biomarkers (MDA and 8-OHdG) at two time measured the expression levels of related gene machineries points that are approximately five years apart (two years and seven (DNMTs, TETs, MBDs, MECP2, and DNA repair genes) to shed light years after the accidental crude oil exposure). It is possible that the on the potential mechanisms responsible for the observed changes relatively small sample size could be responsible for the significant in global DNA methylation levels and other epidemiological differences in measured biomarkers (DNA methylation levels, 5-mc characteristics. and 5-hmc, and oxidative stress biomarkers, MDA and 8-OHdG) As a result, the HSOS-exposed group, compared to the referent based on age, gender and/or smoking habits. group, showed that (i) significant decrease of DNA methylation The expression levels of gene encoding DNA damage repair contents (%5-mc) and ratio of %5-mc and %5-hmc, DNA methylation enzymes are known biomarkers for genetic instability, including gene expression (DNMT3B and MBD1), and increase of DNA epidemiological studies (Collins and Ferguson, 2012). The high demethylation gene expression (TET1 and TET2); (ii) significant substrate specificities of these enzymes make them excellent in- higher expression of DNA repair gene (OGG1, MTH1, ERCC1, XPA) dicators for the type of DNA lesions formed as a result of exposure with decreasing trends over time; (iii) higher levels of the oxidative (Hartwig and Schwerdtle, 2002). The highly significant upregula- stress biomarkers (MDA and 8-OHdG) with increasing trends over tion of NER (ERCC1 and XPA) expression levels and BER (OGG1 gene) time. levels in the AH-baseline and AH-follow-up groups (Table 3, Fig. S3) We could also find that a significant negative correlation be- highlighted the fact that HSOS exposure can cause bulky-DNA tween %5-mc and %5-hmc in the HSOS-exposed with a consistency adduct formation and oxidative DNA lesions (Hartwig and over time, significant positive correlations between 5-mc and Schwerdtle, 2002), which was further corroborated by the persis- DNMT3B and between 5-hmc and TET1, and linear associations tent urinary 8-OHdG formation in the same subjects (Table 3, between oil-cleaning work duration and 5-mc (negatively) and 5- Fig. S1). Crude oil contain Polycyclic aromatic hydrocarbons (PAH) hmc (positively) in the HSOS-exposed. which is known to cause bulky-DNA adduct formation and activa- Previous studies suggested that DNA methylation is highly tion of the NER pathway (Hartwig and Schwerdtle, 2002). sensitive to environmental exposure in both the pre-natal and adult Consistent with previous findings, 5-hmc levels were consid- stages (Cardenas et al., 2017, 2015; De Prins et al., 2013; Fustinoni erably lower than 5-mc levels in the same individuals (Cardenas et al., 2012; Wu et al., 2017). Nonetheless, no studies investigated et al., 2017; Sanchez-Guerra et al., 2015; Tellez-Plaza et al., 2014). the association between crude-oil exposure and DNA methylation Nonetheless, the ratio between 5-mc and 5-hmc levels was levels. To the best of our knowledge, the present study is the first to considerably higher than previously reported values, specifically in investigate associations between both 5-hmc and 5-mc levels and the US Strong heart study, in which the ratio was approximately 2.5 crude-oil exposure. Results revealed highly significant differences fold (Tellez-Plaza et al., 2014), while the order of 5-mc/5-hmc ratios in DNA methylation levels between two time points (2009 vs. (mean values) in our study were AL (137.40) > AH-baseline N. Chatterjee et al. / Environmental Pollution 263 (2020) 114607 7

(93.76) > AH-follow-up (48.75), which were closer to the value 5. Conclusions reported by Sanchez-Guerra et al., which was ~83 fold in Beijing truck drivers exposed to particulate matter less than 10 mm (PM10) Our current findings highlighted the potential use of global 5- (Sanchez-Guerra et al., 2015). Consistent with the above findings, mc and 5-hmc levels as biomarkers for environmental exposure, our 5-hmc results were more similar to those of the Beijing pop- including crude-oil, and supports the idea that both parameters are ulation (Sanchez-Guerra et al., 2015), while our 5-mc results were equally important biomarkers for environmental epidemiologic more similar to those reported in the Beijing population (6.61%) studies. (Sanchez-Guerra et al., 2015) and the Spain group (De Prins et al., 2013). Moreover, adult exposure to arsenic (Tellez-Plaza et al., Ethics approval and consent to participate 2014) and PM10 (Sanchez-Guerra et al., 2015) showed positive correlations with %5-hmc levels, but not with %5-mc levels. On the The study protocol was approved by the Institutional Review other hand, %5-hmc levels showed an inverse association with Board of Dankook University Hospital, and informed consent was prenatal mercury exposure (Cardenas et al., 2017). In contrast to obtained from all subjects (IRB no. 0904-027, 2014-06-013). previous studies, our results showed positive associations of oil cleanup exposure with both 5-mc and 5-hmc contents in the cross- Declaration of competing interest sectional study (AH-baseline) and only with 5-hmc in the longi- tudinal study (Fig. S5). Therefore, our results suggested that both 5- The authors declare no competing interests. mc and 5-hmc levels are important biomarkers and that 5-hmc is likely to be a more sensitive biomarker (Cardenas et al., 2017) for CRediT authorship contribution statement environmental epidemiological studies. The observed positive correlation between crude oil exposure Nivedita Chatterjee: Investigation, Validation, Writing - orig- (as HSOS cleaning works) and 5-hmc levels (Fig. 5, Fig. S6)was inal draft. Jaeseong Jeong: Formal analysis, Visualization, Writing - consistent with the 5-hmc formation cycle. Environmental expo- review & editing. Myung-Sook Park: Data curation. Mina Ha: Re- sure to pro-oxidants can increase the global genomic 5-hmc con- sources, Data curation. Hae-Kwan Cheong: Resources. Jinhee Choi: tents by activating the TET enzyme (TET1 activation, Table 2)(Chia Supervision, Conceptualization. et al., 2011). Persistently elevated 8-OHdG and MDA levels indicate greater oxidative stress (reactive oxygen species, ROS formation) Acknowledgements because of crude oil exposure; in turn, increased ROS levels could induce the oxidation of 5-mc into 5-hmc. However, no significant This work was supported by the Mid-Career Researcher Pro- direct causal relationship was evident between 8-OHdG and 5-hmc gram through the National Research Foundation of Korea (NRF), levels (Fig. S4, Fig. S5), which was most likely caused by direct ROS funded by the Ministry of Science and ICT (NRF- formation, which could be responsible for higher rate of 5-hmc 2017R1A2B3002242) and through the Taean Environmental Health formation and not the adduct 8-OHdG. An association between Center funded by the Ministry of Environment and Government of oxidative stress and global DNA (5-mc) hypomethylation was pre- Taean-gun (Chungchungnam-do). viously reported in chromate manufacturing workers (Wang et al., 2012). Abbreviations The plausible mechanisms by which exposure to crude oil caused alterations in global DNA methylation, 5-mc levels, include Hebei Spirit oil spill HSOS suppression of the expression levels of de novo ‘writer’ enzyme DNA Adult exposed group and survey from 2009 AH-baseline methyltransferase, DNMT3B, and the activation of the expression of Adult exposed group and survey from 2014 AH-follow-up ten-eleven translocation enzyme, TET1 (Table 2), which is respon- Adult low exposed group (reference population) AL sible for the conversion of 5-mc to 5-hmc during DNA demethyla- DNA methylation 5-mc tion (Branco et al., 2012; Brocato and Costa, 2013; Wu and Zhang, DNA hydroxymethylation 5-hmc 2017). DNA methylation levels, including 5-mc and 5-hmc levels, DNA methyltransferase DNMTs regulate chromatin organization and gene expression and therefore Ten-eleven translocation family protein TETs play critical roles in embryonic development, genomic imprinting, methyl-CpG binding domain protein MBDs various diseases, including carcinogenesis, neurodegeneration, and methyl-CpG binding protein 2 MECP2 cardiovascular diseases (Jones, 2012; Koturbash et al., 2011). PAH, Excision repair 1, endonuclease non-catalytic subunit ERCC1 an integral component of HSOS, has been previously reported to Xeroderma pigmentosum complementation group A protein XPA accelerate methylation aging (Li et al., 2018) and also causes global Xeroderma pigmentosum complementation group C protein XPC DNA hypomethylation in cord blood (Herbstman et al., 2009). 8-oxoguanine DNA glycosylase OGG1 Besides, the small samples size as well as missing AL baseline MutT homolog 1 protein MTH1 group, the present study has certain approach-based limitations. 8-hydroxy-2’ edeoxyguanosine 8-OHdG Our investigations were based on only global DNA methylation (5- Malonaldehyde MDA mc) and hydroxymethylation (5-hmc) contents; therefore, it is Reactive-oxygen-species ROS plausible that crude oil exposure exerts differential gene-specific Polycyclic aromatic hydrocarbons PAHs epigenetic effects. However, our findings showed that alterations in 5-mc and 5-hmc levels can serve as epigenetic regulators of Appendix A. Supplementary data increasing carcinogenesis phenomena in crude oil-exposed human population in Taean cohort, Korea. Further studies are required to Supplementary data to this article can be found online at determine whether alterations in 5-mc and 5-hmc levels and their https://doi.org/10.1016/j.envpol.2020.114607. patterns, as observed in the present study, are associated with human health risks, including cancer, within the same population. References

Agarwal, R., Chase, S.D., 2002. Rapid, fluorimetric-liquid chromatographic 8 N. Chatterjee et al. / Environmental Pollution 263 (2020) 114607

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