Stat3 is a candidate epigenetic biomarker of perinatal Bisphenol A exposure associated with murine hepatic tumors with implications for human health

Figures & data

Table 1. Post-natal day 22 (PND22) mouse liver samples (n = 147) and 10-month mouse liver samples (n = 78) for bisulfite sequencing

Age	BPA dose (per kg maternal diet)	Total animals		Animals with hepatic tumors or pre-neoplastic lesions
		Males	Females	Total	Males	Females	Total
PND22 mice	Control	17	20	37
	50 ng	17	31	48
	50 μg	12	10	22
	50 mg	19	21	40
	Total	65	82	147
10-month mice	Control	10	9	19	2	0
	50 ng	10	10	20	1	2
	50 μg	11	10	21	3	2
	50 mg	9	9	18	4	4
	Total	40	38	78	10	8	18

Table 2. Candidate gene regions for bisulfite sequencing

Candidate Gene	Species	Gene Coordinates	RefSeq Isoforms	Assay Coordinates	Assay Genomic Context	CpG island
Esr1	Mouse	Chr10: 5342780-5734495	1	Chr10: 5644941-5645340	Intron	No
ESR1	Human	Chr6: 152128814-152424408	3	Chr6: 152022101-152022700	Intron	No
Il-6st	Mouse	Chr13: 113254278-113297068	1	Chr13: 113294809-113295208	Exon	No
IL-6ST	Human	Chr5: 55230925-55290821	3	Chr5: 55255601-55256200	Intron	Yes
Stat3	Mouse	Chr11: 100748124-100800825	3	Chr11: 100766115-100766514	Exon, plus 100 bp 5′ intron and 150 bp of 3′ intron	Yes
STAT3	Human	Chr17: 40465343-40540513	3	Chr17: 4049190-40492500	Intron	Yes

Figure 1. Stat3 methylation in 10-month mouse livers by dose and tumor status. (A) Average percent DNA methylation at Stat3 by dose group in 10-month mice perinatally exposed to 50 ng (n = 20), 50 µg (n = 21) or 50 mg (n = 18) BPA/kg maternal diet or a phytoestrogen-free control diet (n = 19). (B) Percent DNA methylation at Stat3 by tumor status in 10-month mice perinatally exposed to 50 ng (n = 20), 50 µg (n = 21) or 50 mg (n = 18) BPA/kg maternal diet or a phytoestrogen-free control diet (n = 19). *P <0.1, **P<0.05

Figure 2. Site-specific Stat3 methylation in 10-month mouse livers by dose. (A) Average percent DNA methylation at Stat3 by dose group in 10-month mice perinatally exposed to 50 ng (n = 20), 50 µg (n = 21) or 50 mg (n = 18) BPA/kg maternal diet or a phytoestrogen-free control diet (n = 19). Figure 1A is the same as Figure 2A. These data are repeated for ease of comparison by the reader. (B) Percent DNA methylation at Stat3 by CpG site in 10-month mice perinatally exposed to 50 ng (n = 20), 50 µg (n = 21) or 50 mg (n = 18) BPA/kg maternal diet or a phytoestrogen-free control diet (n = 19). *P < 0.1, **P < 0.05.

Figure 3. Site-specific Stat3 methylation in PND22 mouse livers by dose. (A) Average percent DNA methylation at Stat3 by dose group in post-natal day 22 (PND22) mice perinatally exposed to 50 ng (n = 48), 50 µg (n = 22) or 50 mg (n = 40) BPA/kg maternal diet or a phytoestrogen-free control diet (n = 37). Figure 2A is the same as Figure 1A. These data are repeated for ease of comparison by the reader. (B) Percent DNA methylation at Stat3 by CpG site in PND22 mice perinatally exposed to 50 ng (n = 48), 50 µg (n = 22) or 50 mg (n = 40) BPA/kg maternal diet or a phytoestrogen-free control diet (n = 37). *P < 0.1, **P < 0.05.

Figure 4. STAT3 methylation in human fetal livers by continuous total BPA. Average percent DNA methylation at STAT3 by dose group in human fetal liver samples (n = 50) by continuous liver tissue total BPA.

Figure 5. STAT3 methylation in human fetal livers by continuous free BPA. Average percent DNA methylation at STAT3 by dose group in human fetal liver samples (n = 50) by continuous liver tissue free BPA.

Table 3. Primer sets for bisulfite sequencing.

Assay	Forward Primer	Reverse Primer	Product Size	TA	Number of CpG sites
Esr1	5′-TTGTGTGATTTGGTTAGAATTTGAG-3′	5′- TTAAAAAACTCCCTAACACATTCCC-3′	348 bp	59°C	2
ESR1	5′-GGGAAAAGTTGTATTAAGTTGTATTGTTT-3′	5′-ACTACAACCTCCACCTCCTAAATTC-3′	434 bp	59°C	2
Il-6st	5′-TTTTTTATAGTTGGAGGATTTTGTG-3′	5′- AAACCATACTTCTCTAACAAACCCA-3′	301 bp	59°C	3
IL-6ST	5′-GGAGTATTTTTGGATAAGTTTTTTTGA-3′	5′-AACTCACACCTATAATCCCAATACTTT-3′	381 bp	56°C	7
Stat3	5′-GTTTAGGTAGATGTTGGGAGGGTT-3′	5′-TTCCAAACAAAACATTATAAATAAACTAAC-3′	148 bp	56°C	3
STAT3	5′-ATTAGTATTTGGGAAGGTTGAAGTG-3′	5′- TACAAACATACACCACCAAACTCAA-3′	324 bp	64°C	3

All forward primers contained the following 5′ sequence tag: aggaagagag. All reverse primers contained the following 5′ sequence tag: cagtaatacgactcactatagggagaaggct. T_A refers to annealing temperature.

Table 4. Primer sets for mRNA expression analysis for Stat3, STAT3, and reference genes via qPCR.

Assay	Forward Primer	Reverse Primer
Stat3	5′- AGTTCAAGCACCTGACCCTT -3′	5′-TCAGTCACGATCAAGGAGGC-3′
B2m	5′-CCTTCAGCAAGGACTGGTCT-3′	5′- TCAGTCTCAGTGGGGGTGAA -3′
Gapdh	5′- CCGCATCTTCTTGTGCAGT-3′	5′-GGCAACAATCTCCACTTTGC-3′
STAT3	5′-GGGAAAAGTTGTATTAAGTTGTATTGTTT-3′	5′-ACTACAACCTCCACCTCCTAAATTC-3′
UBC	5′-GATCGCTGTGATCGTCACTT-3′	5′-TCTTTG CCTTGACATTCTCG-3′
GAPDH	5′-CATCAATGGAAATCCCATCA-3′	5′-GACTCCACGACGTACTCAGC-3′