Effects of prenatal exposure to hexafluoropropylene oxide dimer acid on rat and offspring mammary gland development and associated hormone levels.

Elf5 is an epithelial-specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blot analysis revealed that decreased expression of Elf5 correlated with the downregulation of milk proteins in Elf5(+/-) mammary glands. Mammary gland transplants into Rag(-/-) mice demonstrated that Elf5(+/-) mammary alveolar buds failed to develop in an Elf5(+/+) mammary fat pad during pregnancy, demonstrating an epithelial cell autonomous defect. Elf5 expression was reduced in Prolactin receptor (Prlr) heterozygous mammary glands, which phenocopy Elf5(+/-) glands, suggesting that Elf5 and Prlr are in the same pathway. Our data demonstrate that Elf5 is essential for developmental processes in the embryo and in the mammary gland during pregnancy.

Comparative study on gene expression profiles in the liver of male neonatal mice prenatally exposed to PFOA and its alternative HFPO-DA.

Integrin-mediated cell adhesion and signaling is required for mammary gland development and functions. As a major mediator of integrin signaling, focal adhesion kinase (FAK) has been implicated to play a role in the survival, proliferation, and differentiation of mammary epithelial cells in previously studies in vitro. To assess the role of FAK in vivo, we created mice in which FAK is selectively deleted in mammary epithelial cells. The mammary gland FAK conditional knock-out (MFCKO) mice are viable, fertile, and macroscopically indistinguishable from the control littermates. In virgin MFCKO mice, mammary ductal elongation is retarded at 5 weeks of age but reaches the full extent by 8 weeks of age compared with the control mice. However, the MFCKO females are unable to nurse their pups due to severe lobulo-alveolar hypoplasia and secretory immaturity during pregnancy and lactation. Analysis of the mammary epithelial cells in MFCKO mice showed reduced Erk phosphorylation, expression of cyclin D1, and a corresponding decrease in proliferative capability compared with the littermate controls. In addition, phosphorylation of STAT5 and expression of whey acidic protein are significantly reduced in the mammary glands of MFCKO mice, suggesting defective secretory maturation in these mice. Therefore, the combination of the severe lobulo-alveolar hypoplasia and defective secretory differentiation is responsible for the inability of the MFCKO females to nurse their pups. Together, these results provide strong support for a role of FAK in the mammary gland development and function in vivo.

Exposure to perfluorooctanoic acid (PFOA) and hexafluoropropylene oxide dimer acid (HFPO-DA) was associated with adipogenesis. However, potential mechanisms remain to be elucidated. Herein, a 3T3-L1 adipocyte model was used to explore the dynamic changes in adipocyte differentiation (2, 4, and 8 days) under PFOA and HFPO-DA exposure. PFOA and HFPO-DA increased the adipocyte formation rate and intracellular levels of triglycerides (TG). Meanwhile, adipocyte browning was induced by PFOA and HFPO-DA, which was characterized by small lipid droplets, low levels of TG per adipocyte, increased ATP levels, and elevated mitochondrial respiration activities with time-dependent differentiation. The browning potency indexes of PFOA and HFPO-DA were approximately 1.5 times higher than those of the controls. Time-course transcriptomics analysis showed that PFOA and HFPO-DA activated the biological process of adipocyte browning but different gene expression patterns regulated adipocyte browning. Only overexpressed hydroxymethylglutaryl-CoA synthase (Hmgcs2) was shared between PFOA and HFPO-DA groups from 2 to 8 days. Hmgcs2 could regulate adipocyte browning induced by PFOA and HFPO-DA, and this observation was lost when Hmgcs2 was knocked down. Our study suggests that PFOA and HFPO-DA could play dual roles in the differentiation of 3T3-L1 adipocytes, and Hmgcs2 might be a target of PFOA- and HFPO-DA-induced adipocyte browning.

Histone modification alters chromatin architecture and thereby influences gene transcription. Histone methylation status is reversible and counter-regulated by methyltransferases and demethylases. Jmjd1a (also known as KDM3A, TSGA, JMJD1, JHDM2A and JHMD2A) is a histone demethylase. It belongs to JmjC domain-containing protein family and could specifically remove di- and mono- methyl residues from di or mono-methylated histone H3K9 (H3K9me2/me1). Recent studies showed that Jmjd1a plays an important role in embryonic stem cell self-renewal, spermatogenesis, regulation of metabolic gene expression and body weight, sex determination, tumor angiogenesis, and macrophage infiltration. However, its role in mammary gland (MG) development, breast carcinogenesis and breast cancer progression hasn't been systemically investigated. In this study, we found that Jmjd1a is expressed in mouse luminal epithelial cells. Genetic disruption of the Jmjd1a gene significantly slowed down MG development as indicated by retarded MG elongation and decreased ductal density in virgin mice observed at the ages of 4, 6 and 8 weeks. In agreement with the retarded MG development, the expression of Ki67 and cyclinD1 in epithelial cells of MGs from Jmjd1a knockout (KO) mice dramatically reduced compared with that from wild type (WT) mice. H3K9me1 and H3K9me2 levels in the epithelial cells of KO MGs are much higher than that in WT MGs. To assess the role of Jmjd1a in breast cancer progression, we crossbred Tg(Jmjd1a-/-) mice with MMTV-TVA(RCAS-PyMT) mice and obtained Tg(Jmjd1a-/-)×MMTV-TVA(RCAS-PyMT) mice. Infection of the TVA-expressing MG epithelial cells with the RCAS-PyMT virus induced mammary tumors in these mice and MMTV-TVA(RCAS-PyMT) control mice. We found that KO of Jmjd1a slightly accelerated mammary tumor initiation but significantly decreased tumor growth. Ki67 and cyclinD1 expression statistically reduced in KO tumors versus WT tumors. At the molecular level, Jmjd1a expression positively correlated with cyclin D1 expression in mammary epithelial cells and mammary tumors. Knockdown of Jmjd1a in MCF-7 cells significantly reduced cyclin D1 expression, while ectopic expression of Jmjd1a in MCF-7 cells increased cyclin D1 expression. ChIP assay revealed that Jmjd1a is associated with a promoter region of cyclin D1. Co-expression of c-Myc and Jmjd1a boosted the activity of the cyclin D1 reporter. In conclusion, our study indicated that Jmjd1a plays an important role in promoting mammary gland development and breast tumor growth by up-regulating cyclin D1 expression. Targeting Jmjd1a may inhibit breast cancer progression. Citation Format: Qin L, Xu Y, Wu Y, Yu X, Toneff MJ, Liao L, Li Y, Xu J. The role of Jmjd1a in mammary gland development and breast tumor growth. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-04-01.

Lower toxicity of HFPO-DA compared to its predecessor PFOA to the earthworm Eisenia fetida: Evidence from oxidative stress and transcriptomic analysis.

Hirosaki hairless rat (HHR) is a mutant strain spontaneously derived from Sprague-Dawley rats (SDR), and its inheritance is autosomal recessive. In addition to hair loss, female HHRs show involution of the mammary gland at an early stage of lactation. In the present study we investigated the mammary gland development in HHR. Morphological examinations revealed that HHR mammary glands are underdeveloped in virgins and exhibit distended alveoli on day 1 of lactation (L1), followed by involution. Milk secretion was observed on L1 in HHR. Whey acidic protein and other proteins were increased in milk of HHR and heterozygous rats on SDS-polyacrylamide gel electrophoresis. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay revealed apoptosis induction in HHRs at an early stage of lactation. By Western blotting, signal transducer and activator of transcription (STAT) 5A levels in cytoplasmic and nuclear fractions of the mammary glands were not different between HHR and SDR on L1 and L7. Nuclear localization of STAT5A in HHR and SDR was confirmed by immunohistochemistry. Tyr-phosphorylated STAT5A was not detected in HHR but was detected in SDR nuclear fractions. Several proteins modified with O-linked N-acetylglucosamine (O-GlcNAc) were detected in HHR nuclear extract on L1, although not in SDR or heterozygous rats by Western blotting. When HHR nuclear extract was applied to wheat germ agglutinin-agarose, a part of STAT5A was recovered in bound fractions. STAT5A of SDR or heterozygous rat nuclei were not bound to the lectin. Electrophoretic mobility shift assay revealed that STAT5A modified with O-GlcNAc is bound to the STAT5-responsive element. These results indicate that the mammary glands of HHR showed terminal differentiation for a short period, followed immediately by involution. In HHR, STAT5A is modified with O-GlcNAc but is not Tyr-phosphorylated. This type of glycosylation is suggested to be involved in the transient activation of STAT5A in HHR.

Are HFPO-TA and HFPO-DA safe substitutes for PFOA? A comprehensive toxicity study using zebrafish (Danio rerio) embryos and adults

Male mammary gland development and methylation status of estrogen receptor alpha in Wistar rats are modified by the developmental exposure to a glyphosate-based herbicide

Higher toxicity and bioaccumulation of PFOA and HFPO-DA mixture than individuals on the marine model diatom Thalassiosira pseudonana.

Hexafluoropropylene oxide dimer acid [(HFPO-DA), GenX] is listed as Substances of Very High Concern (SVHC) by the Commission of Member States (MSC) in 2019. The threat of GenX to human health, especially to sensitive populations such as pregnant women, infants and children cannot be ignored. We aimed to investigate the effects of GenX on the intestinal barrier function and gut microbiota of both pregnant rats and their offspring. We subsequently performed a metabolomic analysis of the maternal gut to investigate the metabolic disruptions associated with GenX exposure. Pregnant Sprague-Dawley rats (n=12/group) received GenX once daily by gavage at 0, 1, 10, or 100mg/kg body weight from gestational day (GD) 0.5 through GD 19.5. We observed intestinal tissue damage and changes in the gut microbiota in both pregnant rats and their offspring. Additionally, we performed untargeted LC/MS metabolomic analysis on the intestinal tissues of the pregnant rats. The intestinal barrier function of the pregnant rats was impaired. The results of 16S rRNA gene sequencing indicate that GenX alters the gut microbiota diversity and composition in both pregnant rats and their offspring. The metabolites of pregnant rats were also found to be significantly altered, and these metabolites are mainly associated with amino acid metabolism, carbohydrate metabolism, and lipid metabolism. Collectively, Exposure to GenX disrupts the intestinal barrier function of pregnant rats, exacerbates microbiome toxicity, and disturbs the gut microbiome-metabolome homeostasis.

G protein-coupled receptor 120 (GPR120) plays a pivotal role in regulating lactation, yet its underlying mechanisms remain unclear. In mouse models, GPR120 expression in the mammary gland increases markedly during lactation. Under inflammatory conditions, both n-3 polyunsaturated fatty acids (n-3 PUFAs) and GPR120 agonists markedly reduced inflammatory responses and enhanced lipogenesis and migration in HC11 mammary epithelial cells. These benefits were also observed under non-inflammatory conditions and were diminished when GPR120 was knocked down. Furthermore, the regulatory function of GPR120 under non-inflammatory conditions in in vivo and in vitro models is explored. We discovered that the GPR120–Gαs–cyclic adenosine monophosphate (cAMP)–exchange protein directly activated by cAMP (EPAC) signaling axis is critical for lipogenesis and migration in mammary epithelial cells. Through transcriptomic analyses, the EPAC–CCCTC-binding factor (CTCF)–peroxisome proliferator-activated receptor γ (PPARγ)/CCAAT enhancer-binding protein α (C/EBPα) pathway was identified to primarily govern lipogenesis, while the EPAC–C-X-C motif chemokine ligand 14 (CXCL14)/C-X-C chemokine receptor type 4 (CXCR4) autocrine loop regulates migration of mammary epithelial cells. Overall, these findings suggest that GPR120, which can be activated by n-3 PUFAs, improves mammary gland performance by alleviating inflammation and directly modulating mammary lipogenesis and mammary gland development through the CTCF–PPARγ/C/EBPα and CXCL14–CXCR4 pathways. Thus, GPR120 and its downstream signaling targets may represent an important clinical target for enhancing maternal lactation.

Hexafluoropropylene oxide trimer acid (HFPO-TA) and hexafluoropropylene oxide dimer acid (HFPO-DA) have been used as perfluorooctanoic acid (PFOA) alternatives in the fluoropolymer industry for years. Their widespread environmental distribution, high bioaccumulation capability, and human exposure have caused great concern. Nevertheless, their potential toxicity and health risk remain largely unknown. In the present study, we compared potential disruption effects of HFPO-TA, HFPO-DA, and PFOA on peroxisome proliferator-activated receptor γ (PPARγ) via the investigation of receptor binding, receptor activity, and cell adipogenesis effects. The receptor binding experiment showed HFPO-TA exhibited 4.8-7.5 folds higher binding affinity with PPARγ than PFOA, whereas HFPO-DA exhibited weaker binding affinity than PFOA. They also showed agonistic activity toward PPARγ signaling pathway in HEK 293 cells in the order of HFPO-TA > PFOA > HFPO-DA. Molecular docking simulation indicated HFPO-TA formed more hydrogen bonds than PFOA, whereas HFPO-DA formed fewer hydrogen bonds than PFOA. HFPO-TA promoted adipogenic differentiation and lipid accumulation in both mouse and human preadipocytes with potency higher than PFOA. Adipogenesis in human preadipocytes is a more sensitive end point than mouse preadipocytes. Collectively, HFPO-TA exerts higher binding affinity, agonistic activity, and adipogenesis activity than PFOA. The potential health risk of HFPO-TA should be of concern.

Global gene expression and morphological alterations in the mammary gland after gestational exposure to bisphenol A, genistein and indole-3-carbinol in female Sprague-Dawley offspring

Nicotinamide phosphoribosyl transferase in mammary gland epithelial cells is required for nicotinamide mononucleotide production in mouse milk