Preparations of black cohosh extract are sold as dietary supplements marketed to relieve the vasomotor symptoms of menopause, and some studies suggest it may protect against postmenopausal bone loss. Postmenopausal women are also frequently prescribed bisphosphonates, such as risedronate, to prevent osteoporotic bone loss. However, the pharmacodynamic interactions between these compounds when taken together is not known. To investigate possible interactions, 6-month-old, female Sprague-Dawley rats underwent bilateral ovariectomy or sham surgery and were treated for 24 weeks with either vehicle, ethinyl estradiol, risedronate, black cohosh extract or coadministration of risedronate and black cohosh extract, at low or high doses. Bone mineral density (BMD) of the femur, tibia, and lumbar vertebrae was then measured by dual-energy X-ray absorptiometry (DEXA) at weeks 0, 8, 16, and 24. A high dose of risedronate significantly increased BMD of the femur and vertebrae, while black cohosh extract had no significant effect on BMD individually and minimal effects upon coadministration with risedronate. Under these experimental conditions, black cohosh extract alone had no effect on BMD, nor did it negatively impact the BMD-enhancing properties of risedronate.

IntroductionIn 2015, the FDA released a Drug Safety Communication regarding a possible link between opioid exposure during early pregnancy and an increased risk of fetal neural tube defects (NTDs). At the time, the indications for opioid use during pregnancy were not changed due to incomplete maternal toxicity data and limitations in human and animal studies. To assess these knowledge gaps, largescale animal studies are ongoing; however, state-of-the-art technologies have emerged as promising tools to assess otherwise non-standard endpoints. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is a dynamic approach capable of generating 2D ion images to visualize the distribution of an analyte of interest across a tissue section.MethodsGiven the importance of lipid metabolism and neurotransmitters in the developing central nervous system, this study incorporates MALDI MSI to assess lipid distributions across mouse gestational day (GD) 18 fetuses, with and without observable NTDs following maternal exposure on GD 8 to morphine (400 mg/kg BW) or the NTD positive control valproic acid (VPA) (500 mg/kg BW).ResultsAnalysis of whole-body mouse fetuses revealed differential lipid distributions localized mainly in the brain and spinal cord, which included several phosphatidylcholine (PC) species such as PCs 34:1, 34:0, and 36:2 localized to the cortex or hippocampus and lyso PC 16:0 across all brain regions. Overall, differential lipids increased in with maternal morphine and VPA exposure. Neurotransmitter distributions across the brain using FMP-10 derivatizing agent were also assessed, revealing morphine-specific changes.DiscussionThe observed differential glycerophospholipid distributions in relation to treatment and NTD development in mouse fetuses provide potential targets for further investigation of molecular mechanisms of opioid-related developmental effects. Overall, these findings support the feasibility of incorporating MALDI MSI to assess non-standard endpoints of opioid exposure during gestation.

Increased research to improve preclinical models to inform the development of therapeutics for neonatal diseases is an area of great need. This article reviews five common neonatal diseases - bronchopulmonary dysplasia, retinopathy of prematurity, necrotizing enterocolitis, perinatal hypoxic-ischemic encephalopathy and neonatal sepsis - and the available in vivo, in vitro and in silico preclinical models for studying these diseases. Better understanding of the strengths and weaknesses of specialized neonatal disease models will help to improve their utility, may add to the understanding of the mode of action and efficacy of a therapeutic, and/or may improve the understanding of the disease pathology to aid in identification of new therapeutic targets. Although the diseases covered in this article are diverse and require specific approaches, several high-level, overarching key lessons can be learned by evaluating the strengths, weaknesses and gaps in the available models. This Review is intended to help guide current and future researchers toward successful development of therapeutics in these areas of high unmet medical need.

Evidence of structural abnormalities in the nervous system of recreational drug [e.g., phencyclidine (PCP) or ketamine] users and/or preclinical animal research models suggests interference with the activity of multiple neurotransmitters, particularly glutamate neurotransmission. The damage to the central nervous system (CNS) may include neuronal loss, synaptic changes, disturbed neural network formation and reduced projections to subcortical fields. Notably, the reduced projections may considerably compromise the establishment of the subcortical areas, such as the nucleus accumbens located in the basal forebrain. With its abundant dopaminergic innervation, the nucleus accumbens is believed to be directly associated with addictive behaviors and mental disorders. This review seeks to delineate the relationship between PCP/ketamine-induced loss of cortical neurons and the reduced level of polysialic acid neural cell adhesion molecule (PSA-NCAM) in the striatum, and the likely changes in striatal synaptogenesis during development. The basic mechanism of how PSA-NCAM cell surface expression may be regulated will also be discussed, as well as the hypothesis that PSA-NCAM activity is critical to the regulation of synaptic protein expression. Overall, the present review will address the general hypothesis that damage/interruption of cortico-striatal communication and subcortical synaptogenesis could underlie the erratic/sensitization or addictive states produced by chronic or prolonged PCP/ketamine usage.

Birth Defects ResearchVolume 111, Issue 9 p. 405-564 59th Annual Teratology Society MeetingFree Access 59th Annual Teratology Society Meeting First published: 13 May 2019 https://doi.org/10.1002/bdr2.1515AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume111, Issue9Special Issue: 59th Annual Teratology Society MeetingMay 15, 2019Pages 405-564 RelatedInformation

For the past five years, Dr. Daniel Acosta has served as the Deputy Director of Research at the National Center for Toxicological Research (NCTR), a principle research laboratory of the U.S. Food and Drug Administration (FDA). Over his career at NCTR, Dr. Acosta has had a major impact on developing and promoting the use of in vitro assays in regulatory toxicity and product safety assessments. As Dr. Acosta nears his retirement we have dedicated this paper to his many accomplishments at the NCTR. Described within this paper are some of the in vitro studies that have been conducted under Dr. Acosta's leadership. These studies include toxicological assessments involving developmental effects, and the development and application of in vitro reproductive, heart, liver, neurological and airway cell and tissue models.

2-hydroxy-4-methoxybenzophenone (HMB) is an ultraviolet light-absorbing compound that is used in sunscreens, cosmetics and plastics. HMB has been reported to have weak estrogenic activity by in vivo and in vitro studies, making it a chemical with potential reproductive concern. To explore if prenatal and lactational HMB exposure alters gene expression profiles of the developing reproductive organs, we performed microarray analysis using the prostate and testis of postnatal day (PND) 30 male Sprague-Dawley rats offspring exposed to 0, 3000, or 30,000 ppm of HMB from gestational day 6 through PND 21. Gene expression profiles of the prostate and testis were differentially affected by HMB dose with significant alterations observed at the 30,000 ppm HMB group. Tissue-specific gene expression was also identified. These genes, whose expression was altered by HMB exposure, may be considered as candidate biomarker(s) for testicular or prostatic toxicity; however, further studies are necessary to explore this potential.

The mouse embryonic stem cell test (mEST) is a promising in vitro assay for predicting developmental toxicity. In the current study, early differentiation of D3 mouse embryonic stem cells (mESCs) under osteoblast culture conditions and embryotoxicity of cadmium sulfate were examined. D3 mESCs were exposed to cadmium sulfate for 24, 48 or 72 h, and whole genome transcriptional profiles were determined. The results indicate a track of differentiation was identified as mESCs differentiate. Biological processes that were associated with differentiation related genes included embryonic development and, specifically, skeletal system development. Cadmium sulfate inhibited mESC differentiation at all three time points. Functional pathway analysis indicated biological pathways affected included those related to skeletal development, renal and reproductive function. In summary, our results suggest that transcriptional profiles are a sensitive indicator of early mESC differentiation. Transcriptomics may improve the predictivity of the mEST by suggesting possible modes of action for tested chemicals.

In the last several years, a great deal of research has revolved around the refinement of existing alternative tests for developmental toxicity and the development of assays to monitor different endpoints. Differentiation of mouse embryonic stem cells has been used to test the developmental toxicity of a number of compounds. The original test, as validated by the European Center for the Validation of Alternative Methods, was able to correctly categorize 78% of tested chemicals (Genschow et al., 2002). However, refinements and additions to the assay were suggested to increase prediction accuracy. Those suggestions included additional endpoints to evaluate tissues derived from other germ cell layers, more quantifiable endpoints, shorter assay times, and more high-throughput tests. Examples of assays that have addressed each of those concerns are included in this review. Much less work has been done to develop assays to predict developmental toxicity that use human cells. Although the availability of human induced pluripotent stem cells has spurred the development of assays for toxicity testing of other endpoints (e.g., cardiotoxicity), relatively few publications have focused on the use of these cells for developmental toxicity testing. This may be in part because of the difficulty in culturing and differentiating human cells. Examples of some of the assays that have been developed using human cells are also included in this review.

BACKGROUND:The complexity of spermatogenesis makes development of appropriate in vitro testis models challenging. A novel in vitro mouse testis culture system has been reported but not yet evaluated as an alternative model for male reproductive toxicity testing. We assessed the effects of media composition on sperm differentiation and testis morphology of cultured mouse testis fragments. METHODS:Testes from postnatal day 5 B6:CBA-Tg(Acrv1-EGFP)2727Redd/J male mice were cultured in knockout serum replacement (KSR) or Albumax I (Albumax) medium. Enhanced green fluorescent protein (EGFP) expression was examined on days 35, 42, 45, and 49 of culture. Histology and flow cytometry were performed for testis morphology and spermatid differentiation. RESULTS:EGFP signals were first observed in round spermatids on day 22 of culture (corresponding to postnatal day 27) and were observed until the end of culture, indicating testis-specific protein expression. A-kinase anchor protein 4 expression, a marker of elongated spermatid (step 15-16) occurred earlier in explants cultured in KSR than Albumax medium (typically day 35 and after day 42 of culture, respectively). The percentage of seminiferous tubules with elongated spermatid was higher in Albumax than KSR medium from days 45 to 49 of culture. CONCLUSION:Albumax medium may facilitate or support better morphology and spermatid production than KSR medium. Further studies need to improve spermatid production and refinement of this in vitro testis culture system that may be useful as a supplement to current male reproductive toxicity testing or an alternative model in cases where in vivo testing may be unfeasible. Birth Defects Research 109:465-474, 2017. © 2017 Wiley Periodicals, Inc.

Cardiomyocytes derived from human induced pluripotent stem cells are being used for multiple purposes in toxicology. However, currently the phenotype of the cells is similar to that of an immature or fetal cardiomyocyte. Several different methods have been used in an attempt to produce more mature cardiomyocytes; these methods include lengthening the time in culture, electric stimulation, biochemical cues, mechanical stresses, co-culture with other cell types, and altering culture conditions by increasing the stiffness of the substrate or patterning of the substrate. This chapter will review the effects of culture conditions on the maturation of cardiomyocytes.

Birth Defects Research Part A: Clinical and Molecular TeratologyVolume 106, Issue 5 p. 309-456 56th Annual Teratology Society MeetingFree Access 56th Annual Teratology Society Meeting First published: 17 May 2016 https://doi.org/10.1002/bdra.23519AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume106, Issue5Special Issue: 56th Annual Teratology Society MeetingMay 2016Pages 309-456 RelatedInformation

While cyclin dependent kinase 1 (CDK1) has a critical role in controlling resumption of meiosis in oocytes, its role has not been investigated directly in spermatocytes. Unique aspects of male meiosis led us to hypothesize that its role is different in male meiosis than in female meiosis. We generated a conditional knockout (cKO) of the Cdk1 gene in mouse spermatocytes to test this hypothesis. We found that CDK1-null spermatocytes undergo synapsis, chiasmata formation, and desynapsis as is seen in oocytes. Additionally, CDK1-null spermatocytes relocalize SYCP3 to centromeric foci, express H3pSer10, and initiate chromosome condensation. However, CDK1-null spermatocytes fail to form condensed bivalent chromosomes in prophase of meiosis I and instead are arrested at prometaphase. Thus, CDK1 has an essential role in male meiosis that is consistent with what is known about the role of CDK1 in female meiosis, where it is required for formation of condensed bivalent metaphase chromosomes and progression to the first meiotic division. We found that cKO spermatocytes formed fully condensed bivalent chromosomes in the presence of okadaic acid, suggesting that cKO chromosomes are competent to condense, although they do not do so in vivo. Additionally, arrested cKO spermatocytes exhibited irregular cell shape, irregular large nuclei, and large distinctive nucleoli. These cells persist in the seminiferous epithelium through the next seminiferous epithelial cycle with a lack of stage XII checkpoint-associated cell death. This indicates that CDK1 is required upstream of a checkpoint-associated cell death as well as meiotic metaphase progression in mouse spermatocytes.

Silver nano-particles (Ag-NPs) are becoming increasingly prevalent in consumer products as antibacterial agents. The increased use of Ag NP-enhanced products will almost certainly increase environmental silver levels, resulting in increased exposures and the potential for increased adverse reactions including neurotoxic effects. In the present study, embryonic neural stem cells (NSCs) from human and rat fetuses (gestational day-16) were used to determine whether Ag-NPs are capable of causing developmental neurotoxicity. The NSCs were cultured in serum free medium supplemented with appropriate growth factors. On the eighth day in vitro (DIV 8), the cells were exposed to Ag-NPs at concentrations of 1, 5, 10, and 20 μg/ml for 24 h. The cultured cells then were characterized by NSC markers including nestin and SOX2 and a variety of assays were utilized to determine the effects of Ag-NPs on NSC proliferation and viability and the underlying mechanisms associated with these effects. The results indicate that mitochondrial viability (MTT metabolism) was substantially attenuated and LDH release was increased significantly in a dose-dependent manner. Ag-NPs-induced neurotoxicity was further confirmed by up-regulated Bax protein expression, an increased number of TUNEL-positively stained cells, and elevated reactive oxygen species (ROS). NSC proliferation was also significantly decreased by Ag-NPs. Co-administration of acetyl-L-carnitine, an antioxidant agent, effectively blocked the adverse effects associated with Ag-NP exposure.

BACKGROUND2-Hydroxy-4-methoxybenzophenone (HMB) is an ultraviolet (UV) absorbing compound used in many cosmetic products as a UV-protecting agent and in plastics for preventing UV-induced photodecomposition. HMB has been detected in over 95% of randomly collected human urine samples from adults and from premature infants, and it may have estrogenic potential.METHODSTo determine the effects of maternal and lactational exposure to HMB on development and reproductive organs of offspring, time-mated female Harlan Sprague-Dawley rats were dosed with 0, 1000, 3000, 10,000, 25,000, or 50,000 ppm HMB (seven to eight per group) added to chow from gestation day 6 until weaning on postnatal day (PND) 23.RESULTS AND CONCLUSIONExposure to HMB was associated with reduced body and organ weights in female and male offspring. No significant differences were observed in the number of implantation sites/litter, mean resorptions/litter, % litters with resorptions, number and weights of live fetuses, or sex ratios between the control and HMB dose groups. Normalized anogenital distance in male pups at PND 23 was decreased in the highest dose group. Spermatocyte development was impaired in testes of male offspring in the highest dose group. In females, follicular development was delayed in the highest dose group. However, by evaluating levels of the compound in rat serum, the doses at which adverse events occurred are much higher than usual human exposure levels. Thus, exposure to less than 10,000 ppm HMB does not appear to be associated with adverse effects on the reproductive system in rats