Evaluation of the utility of the Beta Human Liver Emulation System (BHLES) for CFSAN's regulatory toxicology program.

Microphysiological systems (MPS), such as organ-on-a-chip platforms, are an emerging alternative model that may be useful for predicting human physiology and/or toxicity. Due to the interest in these platforms, the Center for Food Safety and Applied Nutrition partnered with Emulate to evaluate the utility of the Beta Human Liver Emulation System (BHLES) for its regulatory science program. Using known hepatotoxic compounds (usnic acid, benzbromarone, tamoxifen, and acetaminophen) and compounds that have no reported human cases of liver toxicity (dimethyl sulfoxide, theophylline, and aminohippurate) the platform's performance was evaluated. Chemical toxicity was assessed by albumin secretion, urea and LDH release, nuclei number, mitochondrial membrane potential, and apoptosis. System/platform performance was evaluated in terms of sensitivity and specificity, power, and variability and repeatability. Chemical interactions with the Chip material were also assessed. Preliminary findings suggested that for the model test compounds selected, the BHLES accurately predicted toxicity, demonstrated high sensitivity and specificity, high power, and low variability. However, some compounds interacted with the Chip material indicating variable exposure levels that should be accounted for when planning experimentation. The details of the evaluation are presented herein.

Semi-automated image acquisition and automatic image quantification methods for liver Organ-Chips.

Toxicant exposure can induce acute or chronic alterations in cellular numbers, morphology, and cell function. The quantification of these parameters can provide valuable information regarding a toxicant's effect and/or mechanism of action in organ-on-a-chip toxicity testing platforms. Unfortunately, manual quantification can be variable and time consuming. Additionally, the unique designs of Organ-Chips make automated imaging difficult as current microscopes were not specifically designed for Organ-Chip use. The development of semi-automated and automated imaging and quantification procedures greatly increases the quantity and quality of collected data. Using Emulate's transparent liver Organ-Chip (Liver-Chip) in combination with Keyence's bench-top BZ-X700 All-in-one fluorescence microscope we have developed semi-automated imaging and automated quantification methods for nuclei, mitochondrial viability, and apoptosis. The methods described herein provide alternative imaging options to more costly and space consuming microscopes while still providing necessary features for Organ-Chip evaluation. We were able to detect significant decreases in nuclear number and mitochondrial membrane potential, and significant increases in apoptosis with a model hepatotoxic compound, benzbromarone. These methods have greatly reduced the time and increased the quality of cell number/function data acquisition and demonstrated that these automated quantification methods can detect changes resulting from chemical exposure.

Utilization of a model hepatotoxic compound, diglycolic acid, to evaluate liver Organ-Chip performance and in vitro to in vivo concordance.

Microphysiological systems (MPS) are emerging as potentially predictive models for drug safety and toxicity assessment. To assess the utility of these systems, the Food and Drug Administration partnered with Emulate to evaluate the Human Liver Organ-Chip in a regulatory setting. Diglycolic acid (DGA), a known hepatotoxin, was evaluated in the Liver-Chip and compared to a multi-well plate format to assess the Liver-Chip's capabilities, limitations, overall performance, and concordance with other in vivo and in vitro studies. Cryopreserved primary human hepatocytes were exposed to DGA from 1 to 20 mM in Liver-Chips or traditional multi-well plates. We found that 10 mM or 20 mM of DGA was severely cytotoxic in both platforms, while 5 mM was mildly cytotoxic in Liver-Chips. Additionally, some hepatocyte functions were reduced with 5 mM DGA in Liver-Chips and 1 mM in well plates. Individual well effects were greater or occurred sooner than in the Liver-Chips. Examination of the performance of the Liver-Chip showed that variability was low for biochemical endpoints, but higher for imaging endpoints. Sensitivity and specificity were high. Only 3-4 Liver-Chips were necessary to detect an effect depending on the endpoint and effect size. The specifics of the experiment are found herein.

Effects of Exposure to the Endocrine-Disrupting Chemical Bisphenol A During Critical Windows of Murine Pituitary Development.

Critical windows of development are often more sensitive to endocrine disruption. The murine pituitary gland has two critical windows of development: embryonic gland establishment and neonatal hormone cell expansion. During embryonic development, one environmentally ubiquitous endocrine-disrupting chemical, bisphenol A (BPA), has been shown to alter pituitary development by increasing proliferation and gonadotrope number in females but not males. However, the effects of exposure during the neonatal period have not been examined. Therefore, we dosed pups from postnatal day (PND)0 to PND7 with 0.05, 0.5, and 50 µg/kg/d BPA, environmentally relevant doses, or 50 µg/kg/d estradiol (E2). Mice were collected after dosing at PND7 and at 5 weeks. Dosing mice neonatally with BPA caused sex-specific gene expression changes distinct from those observed with embryonic exposure. At PND7, pituitary Pit1 messenger RNA (mRNA) expression was decreased with BPA 0.05 and 0.5 µg/kg/d in males only. Expression of Pomc mRNA was decreased at 0.5 µg/kg/d BPA in males and at 0.5 and 50 µg/kg/d BPA in females. Similarly, E2 decreased Pomc mRNA in both males and females. However, no noticeable corresponding changes were found in protein expression. Both E2 and BPA suppressed Pomc mRNA in pituitary organ cultures; this repression appeared to be mediated by estrogen receptor-α and estrogen receptor-ß in females and G protein-coupled estrogen receptor in males, as determined by estrogen receptor subtype-selective agonists. These data demonstrated that BPA exposure during neonatal pituitary development has unique sex-specific effects on gene expression and that Pomc repression in males and females can occur through different mechanisms.

Icam5 Expression Exhibits Sex Differences in the Neonatal Pituitary and Is Regulated by Estradiol and Bisphenol A.

Endocrine-disrupting chemicals are prevalent in the environment and can impair reproductive success by affecting the hypothalamic-pituitary-gonadal axis. The developing pituitary gland is sensitive to exposure to endocrine-disrupting chemicals, such as bisphenol A (BPA), and sex-specific effects can occur. However, effects on the critical window of neonatal pituitary gland development in mice have not been explored. Therefore, this study determined baseline gene expression in male and female pituitaries and consequences of environmental exposure to 17ß-estradiol (E2) and BPA on transcription of genes exhibiting sex differences during the neonatal period. Through microarray and quantitative RT-PCR analysis of pituitaries at postnatal day (PND)1, 3 genes were differentially expressed between males and females: Lhb, Fshb, and intracellular adhesion molecule-5 (Icam5). To see whether E2 and BPA exposure regulates these genes, pituitaries were cultured at PND1 with 10(-8) M E2 or 4.4 × 10(-6) M BPA. E2 decreased expression of Lhb, Fshb, and Icam5 mRNA in females but only significantly decreased expression of Icam5 in males. BPA decreased expression of Icam5 similarly to E2, but it did not affect Lhb or Fshb. Importantly, in vivo exposure to 50-µg/kg · d E2 from PND0 to PND7 decreased expression of Lhb, Fshb, and Icam5 mRNA in both males and females, whereas 50-mg/kg · d BPA exposure during the same time frame decreased expression of Icam5 in females only. Overall, we have uncovered that genes differentially expressed between the sexes can be regulated in part by hormonal and chemical signals in vivo and directly at the pituitary and can be regulated in a sex-specific manner.

Analysis of uterine gene expression in interleukin-15 knockout mice reveals uterine natural killer cells do not play a major role in decidualization and associated angiogenesis.

During decidualization, uterine natural killer (uNK) cells are the most abundant immune cell types found in the uterus. Although it is well known that they play key roles in spiral arteriole modification and the maintenance of decidual integrity seen after mid-pregnancy, their roles in the differentiation of decidual cells and accompanying angiogenesis during the process of decidualization is less well characterized. To address this, we used whole-genome Illumina BeadChip analysis to compare the gene expression profiles in implantation segments of the uterus during decidualization on day 7.5 of pregnancy between wild-type and uNK cell-deficient (interleukin-15-knockout) mice. We found almost 300 differentially expressed genes and verified the differential expression of ~60 using quantitative RT-PCR. Notably, there was a lack of differential expression of genes involved in decidualization and angiogenesis and this was also verified by quantitative RT-PCR. Similar endothelial cell densities and proliferation indices were also found in the endometrium between the implantation site tissues of wild-type and knockout mice undergoing decidualization. Overall, the results of this study reveal that uNK cells likely do not play a major role in decidualization and accompanying angiogenesis during implantation. In addition, the study identifies a large number of genes whose expression in implantation-site uterine tissue during decidualization depends on interleukin-15 expression in mice.

Localization of C-Fos-Induced Growth Factor (Figf) mRNA Expression in the Mouse Uterus during Implantation.

The purpose of this study was to characterize the localization of Figf mRNA in the mouse uterus during embryo implantation. Strong Figf mRNA hybridization signals were seen in the primary decidual zone just after the onset of implantation from Days 4.5-6.5. On Day 7.5, this expression continued around the concept us, but in addition we observed high expression of Figf mRNA in the endothelial cells that line the forming vascular sinusoids in the lateral me some trial decidua. Interestingly, on Days 8.5 this high expression continued in the endothelial cells of sinusoids in the lateral me some trial decidual tissue but not in the decidual cells surrounding the concept us. As implantation and placental development finished, Figf mRNA expression remained in the endothelial cells of the sinusoids and spiral arterioles of the decidua basalis. Interestingly, Flt4 mRNA was localized to the endothelial cells lining the sinusoids that form during implantation. Since the endothelial cells of the me some trial sinusoids exhibit a high level of proliferation, we speculate that FIGF-FLT4 signaling may play a role in their formation and function during implantation. This work will provide a basis for further research on the potential role of FIGF-FLT4 signaling in endometrial angiogenesis during implantation in mice.

Tumor necrosis factor receptor subfamily 9 (Tnfrsf9) gene is expressed in distinct cell populations in mouse uterus and conceptus during implantation period of pregnancy.

Tumor necrosis factor receptor subfamily 9 (TNFRSF9) plays a potentially important general role in immune function. Tnfrsf9 gene expression has previously been characterized in late pregnant mouse uterus and placenta. However, little is known about its expression in the uterus during the implantation phase of early pregnancy. We have assessed the levels and localization of Tnfrsf9 expression in the mouse uterus and conceptus during implantation. Relative Tnfrsf9 mRNA levels were significantly higher in implantation than in non-implantation site tissue on days 6.5-8.5 of pregnancy. This increase did not depend on the presence of the conceptus, as mRNA levels were not significantly different between pregnant implantation sites and artificially induced deciduomas. Localization by in situ hybridization revealed a subpopulation of endothelial and uterine natural killer cells expressing Tnfrsf9 in the endometrium during implantation. In the developing conceptus, primary trophoblast giant and ectoplacental cells expressed Tnfrsf9 on days 6.5-8.5, followed by expression in the trophoblast giant cell layers surrounding the conceptus on day 9.5 of pregnancy. Two main splice forms of Tnfrsf9 mRNA exist and encode proteins with distinct biological functions; both mRNA splice forms were present in uterine and conceptus tissues as determined by reverse transcription with the polymerase chain reaction. Thus, both membrane and soluble forms of Tnfrsf9 are expressed in specific cell types of the uterus and conceptus during the progression of implantation in mice and possibly have an important function in this process.