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. 2021 Aug 12;129(8):087003. doi: 10.1289/EHP8795

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EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted.

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Figure 6a is a clustered bar graph titled uppercase f 3 mediobasal hypothalamus, plotting messenger ribonucleic acid (arbitrary units), ranging from 0.0 to 2.5 in increments of 0.5 and 3 to 7 in unit increments (y-axis) across T h, D n m1 m D r d1, and Darp p32 (x-axis) for infant, prepubertal, and adult. Figure 6b is a clustered bar graph titled uppercase f 1 T h p, plotting percent methylation, ranging from 0.0 to 1.0 in increments of 0.2 (y-axis) across C p G sites, ranging from 1 to 13 in unit increments (x-axis). Figure 6c is a bar graph titled uppercase f 1 T h p, plotting percent input, ranging from 0 to 5 in unit increments (y-axis) across H3K27m e3, H3K9m e3, H3K4m e3, and H3K9a c, including control and endocrine disrupting chemicals. Figure 6d is a set of two line graphs, plotting uppercase y (micrometer), ranging from 300 to 0 in decrements of 50 (y-axis) across y (micrometer), ranging from 0 to 800 in increments of 100 (x-axis) for control, T H, mPoA, and third ventricle; and Endocrine Disrupting Chemicals, T H, mPoA, and third ventricle, respectively. Figure 6e is a bar graph, plotting T H-ir cells per cubic millimeter, ranging from 0 to 200 in increments of 50 (left y-axis) and T H-ir cells per cubic millimeter, ranging from 0 to 5 in unit increments (right y-axis) across control, endocrine disrupting chemicals, control, endocrine disrupting chemicals, control, and endocrine disrupting chemicals for S N, V T A, and mPoA. Figure 6f is a clustered bar graph titled uppercase f 3 mediobasal hypothalamus, plotting messenger ribonucleic acid (arbitrary units), ranging from 0.0 to 1.5 in increments of 0.5 (y-axis) across T H and D r d1 (x-axis) for infant, prepubertal, and adult. Figure 6g is a clustered bar graph titled uppercase f 3 T h p, plotting percent methylation, ranging from 0.0 to 1.0 in increments 0.2 (y-axis) across C p G site, ranging from 1 to 13 in unit increments (x-axis) for control and endocrine disrupting chemicals. Figure 6h is a bar graph titled uppercase f 3 T h p, plotting percent input, ranging from 0 to 6 in unit increments (y-axis) across H3K27m e3, H3K9m e3, H3K4m e3, and H3K9a c, including control and endocrine disrupting chemicals. — Dopaminergic signaling proteins, mRNA expression and chromatin state in the female rat in utero (F1 generation) and ancestrally (F3 generation) exposed to EDC mixture or vehicle. (A) Expression of Th, Dnm1, Drd1, and Darpp32 mRNA in the MBH-PoA of infant (P6), prepubertal (P21) and adult (P60) F1 female as determined by qPCR ( $n = 6 / group$ ). Samples originates from six different litters per group. RNA expression data were normalized by dividing each individual value by the control group average value at every time point. (B) Methylation state at 13 CpG sites of the Th gene promoter from MBH-PoA explants of F1 females at P21 ( $n = 6 / group$ ). Samples originates from six different litters per group. (C) Abundance of the TrxG-dependent activating marks H3K4me3 and H3K9ac and the PcG-dependent repressive mark H3K27me3 and H3K9me3 at the Th promoter in the prepubertal MBH-PoA of F1 females, as measured by ChIP ( $n = 6 / group$ ). Samples originate from 6 different litters per group. (D) Abundance of Th-immunoreactive cells (green) within the mPoA of F1 prepubertal female rats. (E) Quantification of Th immunoreactivity in the SN, VTA, and mPoA of F1 females. Bars represent mean number of cells per $cubic millimeter \pm SEM$ ; (F) Expression of Th and Drd1 mRNA in the MBH-PoA of infant (P6), prepubertal (P21), and adult (P60) F3 females ( $n = 6 / group$ ). Samples originates from 6 different litters per group. (G) Methylation state at 13 CpG sites of the Th gene promoter from MBH-PoA explants of F3 females at P2 ( $n = 6 / group$ ) Samples originates from six different litters per group. (H) Abundance of H3K4me3, H3K9ac, H3K27me3, and H3K9me3 histone posttranslational modifications at the Th promoter in the prepubertal MBH-PoA of F3 females, as measured by ChIP ( $n = 6 / group$ ). Dotted red lines represent repressive histone modifications (H3K27me3 and H3K9me3). Green solid lines represent activatory histone modifications (H3K27me3 and H3K9ac). EDC data from panels A, C, F, and H were normalized to control data. Samples originates from six different litters per group. Bars represent $mean \pm SEM$ (* $p < 0.05$ , ** $p < 0.01$ , *** $p < 0.001$ vs. CTL). Data was analyzed using either two-way ANOVA (6A), followed by Sidak’s multiple comparisons test or Student’s $t$ -test (6B–H). Summary data are reported in Table S3. Note: 3V, third ventricle; ANOVA, analysis of variance; AU, arbitrary units; ChIP, chromatin immunoprecipitation; CTL, control; EDC, endocrine-disrupting chemical; SN, substantia nigra; Th, tyrosine hydroxylase; VTA, ventral tegmental area.