Decreased global DNA hydroxymethylation in neural tube defects: Association with polycyclic aromatic hydrocarbons

Figures & data

Table 1. Demographic and obstetric characteristics of neural tube defect cases and controls in Shanxi Province, China, 2011–2014.

Characteristic	Cases^a	Controls^a	p^b
Maternal age (y)			0.591
<25	34 (44.2)	16 (55.2)
25–29	21 (27.3)	6 (20.7)
≥30	22 (28.6)	7 (24.1)
BMI (kg/m^2)
<18.5	8 (10.7)	2 (6.5)	0.608
18.5–24.9	41 (54.7)	20 (64.5)
≥25	26 (34.7)	9 (29.0)
Maternal education			0.002
Primary school or less	9 (9.8)	4 (12.1)
Junior high	66 (71.7)	13 (39.4)
High school or more	17 (18.5)	16 (48.5)
Occupation			<0.001
Farmer	75 (83.3)	16 (48.5)
Non-farmer	15 (16.7)	17 (51.5)
Previous history of birth defects			0.324
Yes	5 (5.0)	0 (0.0)
No	87 (95.0)	33 (100.0)
Gravidity			0.094
1	37 (40.7)	19 (57.6)
≥2	54 (59.3)	14 (42.4)
Parity			0.013
1	44 (50.6)	25 (75.8)
≥2	43 (49.6)	8 (24.2)
Periconceptional folic acid supplementation			0.001
Yes	53 (58.2)	8 (24.2)
No	38 (41.8)	25 (75.8)
Fever or flu during early pregnancy			0.088
Yes	31 (34.1)	6 (18.2)
No	60 (65.9)	27 (81.8)
Active or passive smoking			0.170
Yes	54 (59.8)	14 (48.5)
No	37 (40.2)	17 (51.5)
Alcohol drinking			1.000
Yes	5 (5.4)	1 (3.0)
No	87 (94.6)	32 (97.0)
Gestational age (weeks)			0.180
13–27	63 (70.0)	17 (56.7)
≥28	27 (30.0)	13 (43.3)
Sex of fetus			0.155
Male	41 (48.2)	19 (63.3)
Female	44 (51.8)	11 (36.7)

BMI, body mass index. ^aData are frequency (percentage). The total may not equal the total number of cases or controls because of missing or unknown data. ^bCases and controls were compared using Pearson’s chi-square test or Fisher’s exact test if any cell was expected to be less than 5.

Table 2. Levels of 5-hydroxymethylcytosine% (5hmC/total DNA) in fetal neural tissues of NTD cases and controls in Shanxi Province, China, 2011–2014.

Subject	n	Median (IQR)^a	p^b
Total NTDs	92	0.25 (0.12–0.39)	0.003
Anencephaly	39	0.23 (0.085–0.50)	0.018
Spina bifida	45	0.25 (0.16–0.34)	0.014
Encephalocele	8	0.10 (0.04–0.36)	0.016
Controls	33	0.45 (0.19–1.00)	-

NTD, neural tube defect; IQR, interquartile range. ^aData are percentages. ^bCompared to the median of the controls, Mann–Whiney U test.

Table 3. Tertiles of 5-hydroxymethylcytosine% (5hmC/total DNA) in fetal neural tissues and risk for NTDs in Shanxi Province, China, 2011–2014.

5hmC%^b	No. of controls	No. of NTD cases	Unadjusted OR (95% CI)	Adjusted OR (95% CI)^a
<0.281	11	54	7.71 (2.45, 24.32)	7.89 (2.32, 26.86)
0.281–0.840	11	31	4.43 (1.37, 14.28)	4.51 (1.29, 15.72)
>0.840	11	7	1	1
p for trend			0.001	0.002

NTD, neural tube defect; OR, odds ratio; CI, confidence interval. ^aAdjusted for periconceptional folate supplementation. ^bTertiles of total 5hmC% in controls were used as cutoff values.

Table 4. Correlations between concentrations of PAHs in fetal liver tissues and 5hmC% in fetal neural tissues in NTD cases^a.

PAHs	Unadjusted		Adjusted^b
	β (95% CI)	p	β (95% CI)	p
H_PAH	−0.002 (−0.003, 0.000)	0.005	−0.002 (−0.003, 0.000)	0.014
L_PAH	0.000 (0.000, 0.001)	0.045	0.000 (0.000, 0.001)	0.065

PAH, polycyclic aromatic hydrocarbon; NTD, neural tube defect; CI, confidence interval; H_PAH, high-molecular-weight PAH; L_PAH, low-molecular-weight PAH. ^aA generalized linear regression model was used to estimate the association between PAHs and 5hmC% in NTD cases (n = 62). ^bAdjusted for periconceptional folic acid supplementation.

Figure 1. Quantification of 5hmC% in the neural tissue of E10.5 embryos exposed to benzo(a)pyrene (BaP) using an ELISA-like assay (mean±SD; n = 12).

DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay. *p < 0.05 vs. DMSO control.

Figure 2. Reactive oxygen species in E9.5 embryos exposed to BaP.

(A) Reactive oxygen species in embryos measured with whole-mount staining for CMH2DCFDA after 24 h of in vitro whole-embryo culture (mean±SD; n = 12–17). (B) Representative images of reactive oxygen species measured with whole-mount staining for CMH2DCFDA in E9.5 mouse embryos. i: control, ii: BaP. DMSO, dimethyl sulfoxide; BaP, benzo(a)pyrene. *p < 0.05 vs. DMSO control.