Fetal Alcohol Exposure Reduces Dopamine Receptor D2 and Increases Pituitary Weight and Prolactin Production via Epigenetic Mechanisms

Abstract

Recent evidence indicated that alcohol exposure during the fetal period increases the susceptibility to tumor development in mammary and prostate tissues. Whether fetal alcohol exposure increases the susceptibility to prolactin-producing tumor (prolactinoma) development in the pituitary was studied by employing the animal model of estradiol-induced prolactinomas in Fischer 344 female rats. We employed an animal model of fetal alcohol exposure that simulates binge alcohol drinking during the first two trimesters of human pregnancy and involves feeding pregnant rats with a liquid diet containing 6.7% alcohol during gestational day 7 to day 21. Control rats were pair-fed with isocaloric liquid diet or fed ad libitum with rat chow diet. Adult alcohol exposed and control female offspring rats were used in this study on the day of estrus or after estrogen treatment. Results show that fetal alcohol-exposed rats had increased levels of pituitary weight, pituitary prolactin (PRL) protein and mRNA, and plasma PRL. However, these rats show decreased pituitary levels of dopamine D2 receptor (D2R) mRNA and protein and increased pituitary levels of D2R promoter methylation. Also, they show elevated pituitary mRNA levels of DNA methylating genes (DNMT1, DNMT3b, MeCP2) and histone modifying genes (HDAC2, HDAC4, G9a). When fetal alcohol exposed rats were treated neonatally with a DNA methylation inhibitor 5-Aza deoxycytidine and/or a HDAC inhibitor trichostatin-A their pituitary D2R mRNA, pituitary weights and plasma PRL levels were normalized. These data suggest that fetal alcohol exposure programs the pituitary to increase the susceptibility to the development of prolactinomas possibly by enhancing the methylation of the D2R gene promoter and repressing the synthesis and control of D2R on PRL-producing cells.

Fig 1. Effects of fetal alcohol exposure on pituitary weight, plasma PRL and percentage of mitotic lactotropes in pituitaries of cyclic, ovariectomized and estrogen-treated ovariectomized animals.

Alcohol-fed (AF), pair-fed (PF) or ad libitum-fed (AD) rat offsprings were used during the adult period (75–80 days) either on the day of estrus (cyclic) or 15 days after ovariectomy and empty implants (OVEX) or β-estradiol implants (E2-15 d). Mean ± SEM values of pituitary weight (A) and plasma PRL (B) in cyclic animals, pituitary weight (C) and plasma PRL (D) in OVEX or E2-15 d animals, and the percentage of mitotic lactotropes in pituitary of cyclic (E) and E2-15 d (F) animals are shown in the histograms. Data are mean ± SEM of n = 6–8 animals per each group and were analyzed using one-way ANOVA with the Newman-Keuls post hoc test. **, P<0.01 and ***, P<0.001 between AF and controls (AD or PF).

Fig 2. Effect of fetal alcohol exposure on the sensitivity of pituitary lactotropes to the estrogen tumor-promoting action.

Alcohol-fed (AF) or control-fed (PF, AD) rats offspring were ovariectomized and implanted with a β-estradiol implants at 60 days of age and after 60 (E2-60 d) or 90 days (E2-90 d) were used for this study. Mean ± SEM values of pituitary weight at 60 d (A) and 90 d (B), pituitary PRL mRNA level at 60 d (C) and 90 d (D), pituitary PRL protein level at 60 d (E) and 90 d (F), and plasma PRL levels at 60 d (G) and 90 d (H) are shown in the histograms. Data are mean ± SEM of n = 4–8 animals per group and were analyzed using one-way ANOVA with the Newman-Keuls post hoc test. *, P<0.05 and ***, P<0.001 between AF and controls (AD or PF).

Fig 3. Effect of fetal alcohol exposure on D2R expression in the pituitary.

D2R mRNA (A, B) or D2R protein levels (C, D) in pituitaries of AD, PF, AF rat offspring were measured after ovariectomy and 60 or 90 d of estrogen implants, respectively. Data are mean ± SEM of n = 6 animals per each group and were analyzed using one-way ANOVA with the Newman-Keuls post hoc test. *, P<0.05, **, P<0.01, and ***, P<0.001 between AF and controls (AD or PF).

Fig 4. Effect of fetal alcohol exposure on D2R promoter methylation in the pituitary.

A. Schematic representation of rat D2R promoter CpG island. Rat D2R promoter sequence was analyzed to search for CpG islands by Methyl primer v1 program. Each small vertical line represents single CpG. The thick solid bar below represents a CpG island. Arrows represent primer annealing sites (A). D2R promoter methylation in pituitaries of AD, PF, AF rats after ovariectomized and estrogen treatment for 60 or 90 days was performed by real time methylation specific PCR. Methylation was measured as ratio of methylated verses unmethylated DNA. D2R methylation in pituitary of AD, PF, AF rats after ovariectomized and estrogen treatment for 60 (B) and 90 days (C). Pyrosequencing analysis of 3 individual CpGs in the CpG island of the D2R promoter in pituitaries of AD, PF, AF rats after ovariectomized and estrogen treated for 60 days (D). Data are mean ± SEM of n = 6 animals per each group and were analyzed using one-way ANOVA with the Newman-Keuls post hoc test. **, P<0.01 between AF and control AD or PF.

Fig 5. Effect of fetal alcohol exposure on the level of genes regulating DNA methylation and histone modification in the pituitary.

Pituitary mRNA levels of DNMT1 (A), DNMT3a (B), DNMT3b (C), MeCP2 (D), HDAC2 (E), HDAC4 (F), G91 (G) and Set7 (H) of AD, PF. AF rats after ovariectomy and estrogen treatment for 60 days. Data are mean ± SEM of n = 6 animals per group and were analyzed using one-way ANOVA with the Newman-Keuls post hoc test. *, P<0.05, **, P<0.01, and ***, P<0.001 between AF and controls (AD or PF).

Fig 6. Effect of fetal alcohol exposure on DNA methylation and histone deacetylation proteins in the pituitary.

Pituitary protein levels of DNMT1 (A), DNMT3b (B), HDAC2 (C), HDAC4 (D) of AD, PF and AF rats after ovariectomy and estrogen treatment for 60 days. Data are mean + SEM of n = 6 animals per group and were analyzed using one-way ANOVA with the Newman-Keuls post hoc test. *, P<0.05, **, P<0.01 between AF and controls (AD or PF).

Fig 7. Effects of epigenetic modulatory drugs on fetal alcohol induced changes in pituitary D2R mRNA levels, protein levels, pituitary weight and plasma PRL levels.

AD, PF and AF rat offspring were treated with DNA methyl transferase inhibitor 5-Aza deoxycytidine (5-AZAdC), HDACC inhibitor Trichostatin-A (TSA) alone or together during postnatal day 2–6 and after 60 days, these rats were ovariectomized and estrogen treated for 60 days. Mean ± SEM values of pituitary D2R mRNA levels (A), pituitary weight (B) and plasma PRL levels (C) are shown in the histograms. Data are mean ± SEM of n = 6 animals per group and were analyzed using one-way ANOVA with the Newman-Keuls post hoc test. *, P<0.05, between AF and controls (AD or PF). @, P<0.05 between the treatment and control in AF group.

Fig 8. Schematic diagram illustrating fetal alcohol exposure induced epigenetic changes regulating D2R expression and its control of PRL synthesis and lactotropic cell growth in the pituitary.

In control rats, D2R mRNA expression is regulated by the epigenetic mechanism involving DNA demethylation and histone deacetylation of the D2R promoter. D2R participates in mediating the inhibitory action of dopamine on PRL synthesis and cell proliferation in lactotropic cells of the pituitary gland (A). In fetal alcohol exposed (FAE) rats, increased D2R promoter methylation and histone deacetylation result in reduced D2R expression, The lower number of D2Rs prevents dopamine to act on lactotropes causing more PRL production and increased cell proliferation (B). DNA wrapped with histone octomer is represented as black thread with cylindrical structures. Methylated CpG is represented as pentagon structure in the DNA. Acetyl groups of histones are represented as triangles and methyl groups as pentagons on N terminal tails of histone.