Obesogenic Diet in Mice Leads to Inflammation and Oxidative Stress in the Mother in Association with Sex-Specific Changes in Fetal Development, Inflammatory Markers and Placental Transcriptome.

BACKGROUND: Obesity during pregnancy is related to adverse maternal and neonatal outcomes. Factors involved in these outcomes may include increased maternal insulin resistance, inflammation, oxidative stress, and nutrient mishandling. The placenta is the primary determinant of fetal outcomes, and its function can be impacted by maternal obesity. The aim of this study on mice was to determine the effect of obesity on maternal lipid handling, inflammatory and redox state, and placental oxidative stress, inflammatory signaling, and gene expression relative to female and male fetal growth. METHODS: Female mice were fed control or obesogenic high-fat/high-sugar diet (HFHS) from 9 weeks prior to, and during, pregnancy. On day 18.5 of pregnancy, maternal plasma, and liver, placenta, and fetal serum were collected to examine the immune and redox states. The placental labyrinth zone (Lz) was dissected for RNA-sequencing analysis of gene expression changes. RESULTS: the HFHS diet induced, in the dams, hepatic steatosis, oxidative stress (reduced catalase, elevated protein oxidation) and the activation of pro-inflammatory pathways (p38-MAPK), along with imbalanced circulating cytokine concentrations (increased IL-6 and decreased IL-5 and IL-17A). HFHS fetuses were asymmetrically growth-restricted, showing sex-specific changes in circulating cytokines (GM-CSF, TNF-α, IL-6 and IFN-γ). The morphology of the placenta Lz was modified by an HFHS diet, in association with sex-specific alterations in the expression of genes and proteins implicated in oxidative stress, inflammation, and stress signaling. Placental gene expression changes were comparable to that seen in models of intrauterine inflammation and were related to a transcriptional network involving transcription factors, LYL1 and PLAG1. CONCLUSION: This study shows that fetal growth restriction with maternal obesity is related to elevated oxidative stress, inflammatory pathways, and sex-specific placental changes. Our data are important, given the marked consequences and the rising rates of obesity worldwide.

Placental inflammation, oxidative stress, and fetal outcomes in maternal obesity.

The obesity epidemic has led to a growing body of research investigating the consequences of maternal obesity on pregnancy and offspring health. The placenta, traditionally viewed as a passive intermediary between mother and fetus, is known to play a critical role in modulating the intrauterine environment and fetal development, and we now know that maternal obesity leads to increased inflammation, oxidative stress, and altered placental function. Here, we review recent research exploring the involvement of inflammation and oxidative stress as mechanisms impacting the placenta and fetus during obese pregnancy. Understanding them is crucial for informing strategies that can mitigate the adverse health effects of maternal obesity on offspring development and disease risk.

Clofazimine pharmacokinetics in HIV-infected adults with diarrhea: Implications of diarrheal disease on absorption of orally administered therapeutics.

Oral drug absorption kinetics are usually established in populations with a properly functioning gastrointestinal tract. However, many diseases and therapeutics can alter gastrointestinal physiology and cause diarrhea. The extent of diarrhea-associated impact on drug pharmacokinetics has not been quantitatively described. To address this knowledge gap, we used a population pharmacokinetic modeling approach with data collected in a phase IIa study of matched human immunodeficiency virus (HIV)-infected adults with/without cryptosporidiosis and diarrhea to examine diarrhea-associated impact on oral clofazimine pharmacokinetics. A population pharmacokinetic model was developed with 428 plasma samples from 23 HIV-infected adults with/without Cryptosporidium infection using nonlinear mixed-effects modeling. Covariates describing cryptosporidiosis-associated diarrhea severity (e.g., number of diarrhea episodes, diarrhea grade) or HIV infection (e.g., viral load, CD4+ T cell count) were evaluated. A two-compartment model with lag time and first-order absorption and elimination best fit the data. Maximum diarrhea grade over the study duration was found to be associated with a more than sixfold reduction in clofazimine bioavailability. Apparent clofazimine clearance, intercompartmental clearance, central volume of distribution, and peripheral volume of distribution were 3.71 L/h, 18.2 L/h (interindividual variability [IIV] 45.0%), 473 L (IIV 3.46%), and 3434 L, respectively. The absorption rate constant was 0.625 h-1 (IIV 149%) and absorption lag time was 1.83 h. In conclusion, the maximum diarrhea grade observed for the duration of oral clofazimine administration was associated with a significant reduction in clofazimine bioavailability. Our results highlight the importance of studying disease impacts on oral therapeutic pharmacokinetics to inform dose optimization and maximize the chance of treatment success.

Potential and Challenges in Application of Physiologically Based Pharmacokinetic Modeling in Predicting Diarrheal Disease Impact on Oral Drug Pharmacokinetics.

Physiologically based pharmacokinetic (PBPK) modeling is a physiologically relevant approach that integrates drug-specific and system parameters to generate pharmacokinetic predictions for target populations. It has gained immense popularity for drug-drug interaction, organ impairment, and special population studies over the past two decades. However, an application of PBPK modeling with great potential remains rather overlooked - prediction of diarrheal disease impact on oral drug pharmacokinetics. Oral drug absorption is a complex process involving the interplay between physicochemical characteristics of the drug and physiological conditions in the gastrointestinal tract. Diarrhea, a condition common to numerous diseases impacting many worldwide, is associated with physiological changes in many processes critical to oral drug absorption. In this review, we outline key processes governing oral drug absorption, provide a high-level overview of key parameters for modeling oral drug absorption in PBPK models, examine how diarrheal diseases may impact these processes based on literature findings, illustrate the clinical relevance of diarrheal disease impact on oral drug absorption, and discuss the potential and challenges of applying PBPK modeling in predicting disease impacts. Significance Statement Statement Pathophysiological changes resulting from diarrheal diseases can alter important factors governing oral drug absorption, contributing to suboptimal drug exposure and treatment failure. Physiologically based pharmacokinetic (PBPK) modeling is an in silico approach that has been increasingly adopted for drug-drug interaction potential, organ impairment, and special population assessment. This minireview highlights the potential and challenges of using PBPK modeling as a tool to improve our understanding of how diarrheal diseases impact oral drug pharmacokinetics.

Vitamin D and Calcium Supplement Use and High-Risk Breast Cancer: A Case-Control Study among BRCA1 and BRCA2 Mutation Carriers.

The role of vitamin D and calcium use in the development of breast cancer among women in the general population is not clear. Furthermore, whether vitamin D and calcium supplement use are associated with breast cancer in high-risk populations has not been evaluated. Thus, we evaluated the association between vitamin D and/or calcium supplement use and breast cancer among women with a pathogenic variant (mutation) in BRCA1 or BRCA2. BRCA mutation carriers enrolled in a longitudinal study were invited to complete a supplemental questionnaire on lifetime supplement use. Cases included women with a prevalent diagnosis of invasive breast cancer, and controls had no history of breast cancer. Vitamin D and calcium use were categorized as never/ever use, and as tertiles of supplement intake (total average daily supplement use). Unconditional logistic regression was used to estimate the odds ratio (OR) and 95% confidence intervals (CIs) of breast cancer. This study included 134 breast cancer cases and 276 controls. Women who used vitamin D-containing supplements had 46% lower odds of having breast cancer compared to those who never used supplements (OR 0.54; 95% CI 0.31, 0.91; p = 0.02). Increasing vitamin D and calcium supplement intake was inversely associated with the odds of having breast cancer (p-trend = 0.04). Findings were suggestively stronger among BRCA1 mutation carriers; however, analyses were limited by small strata. These findings suggest a potential inverse association between vitamin D and calcium supplementation and BRCA breast cancer. Additional studies are warranted to confirm these findings and accurately inform clinical care guidelines.

Evaluating depression and anxiety throughout pregnancy and after birth: impact of the COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic presents unique social, economic, and psychological challenges for individuals globally. Thus, women who are pregnant face unprecedented mental health challenges. OBJECTIVE: We sought to determine the impact of the pandemic on perinatal depression and anxiety in a longitudinal pregnancy cohort. We hypothesized increased depression and anxiety scores in women during pregnancy and after birth in the pandemic at all time points. STUDY DESIGN: Participants were enrolled in the Ontario Birth Study, a pregnancy cohort embedded in clinical care at Mount Sinai Hospital, Toronto, Canada. Perinatal depression and anxiety were assessed using the 2-Item Patient Health Questionnaire and 2-Item Generalized Anxiety Disorder Questionnaire in early pregnancy, whereas the Edinburgh Postnatal Depression Scale and 2-Item Generalized Anxiety Disorder Questionnaire were used in late pregnancy and after birth. Logistic regression models were created to examine the association of the pandemic with clinically elevated mental health scores in the prepandemic group vs pandemic group while adjusting for covariates. RESULTS: A total of 1159 survey responses from 649 participants between March 1, 2019, and February 28, 2021, were used to conduct this study. Participants were assessed in early pregnancy (n=416), in late pregnancy (n=373), and after birth (n=370). Responses received on or before February 29, 2020, were considered the "prepandemic" responses, whereas responses after the aforementioned date were considered the "pandemic" responses. Mean rank scores of depression and anxiety were significantly higher in the pandemic group (P=.02 and P=.003, respectively) in the postpartum period. There was no significant association between pandemic time and antenatal scores. However, postnatally, mothers were 2.6 times more likely to score ≥13 on the Edinburgh Postnatal Depression Scale during the pandemic than before the pandemic (95% confidence interval, 1.2-5.7; P=.02). Adjustment for ethnicity and income strengthened this association as the odds ratio increased to 3.3 (95% confidence interval, 1.4-8.0; P=.007). CONCLUSION: Pandemic-associated increases in depression and anxiety scores were confined to the postpartum period, highlighting a need for increased screening and interventions for perinatal mood and anxiety disorders postnatally as this pandemic continues.

Pharmacokinetics and Pharmacodynamics of Clofazimine for Treatment of Cryptosporidiosis.

Infection with Cryptosporidium spp. can cause severe diarrhea, leading to long-term adverse impacts and even death in malnourished children and immunocompromised patients. The only FDA-approved drug for treating cryptosporidiosis, nitazoxanide, has limited efficacy in the populations impacted the most by the diarrheal disease, and safe, effective treatment options are urgently needed. Initially identified by a large-scale phenotypic screening campaign, the antimycobacterial therapeutic clofazimine demonstrated great promise in both in vitro and in vivo preclinical models of Cryptosporidium infection. Unfortunately, a phase 2a clinical trial in HIV-infected adults with cryptosporidiosis did not identify any clofazimine treatment effect on Cryptosporidium infection burden or clinical outcomes. To explore whether clofazimine's lack of efficacy in the phase 2a trial may have been due to subtherapeutic clofazimine concentrations, a pharmacokinetic/pharmacodynamic modeling approach was undertaken to determine the relationship between clofazimine in vivo concentrations and treatment effects in multiple preclinical infection models. Exposure-response relationships were characterized using Emax and logistic models, which allowed predictions of efficacious clofazimine concentrations for the control and reduction of disease burden. After establishing exposure-response relationships for clofazimine treatment of Cryptosporidium infection in our preclinical model studies, it was unmistakable that the clofazimine levels observed in the phase 2a study participants were well below concentrations associated with anti-Cryptosporidium efficacy. Thus, despite a dosing regimen above the highest doses recommended for mycobacterial therapy, it is very likely the lack of treatment effect in the phase 2a trial was at least partially due to clofazimine concentrations below those required for efficacy against cryptosporidiosis. It is unlikely that clofazimine will provide a remedy for the large number of cryptosporidiosis patients currently without a viable treatment option unless alternative, safe clofazimine formulations with improved oral absorption are developed. (This study has been registered in ClinicalTrials.gov under identifier NCT03341767.).

Habitual Intake of Marine-Derived n-3 PUFAs is Inversely Associated with a Cardiometabolic Inflammatory Profile in Yup'ik Alaska Native People.

BACKGROUND: The relationship between dietary n-3 PUFAs and the prevention of cardiometabolic diseases, including type 2 diabetes, is unresolved. Examination of the association between n-3 PUFAs and chronic low-grade inflammation in a population where many individuals have had an extremely high intake of marine mammals and fish throughout their lifespan may provide important clues regarding the impact of n-3 PUFAs on health. OBJECTIVES: The aim of this study was to explore associations between concentrations of n-3 PUFAs resulting from habitual intake of natural food sources high in fish and marine mammals with immune biomarkers of metabolic inflammation and parameters of glucose regulation. METHODS: A total of 569 Yup'ik Alaska Native adults (18-87 years old) were enrolled in this cross-sectional study between December 2016 and November 2019. The RBC nitrogen isotope ratio (NIR; 15N/14N) was used as a validated measure of n-3 PUFA intake to select 165 participant samples from the first and fourth quartiles of n-3 PUFA intakes. Outcomes included 38 pro- and anti-inflammatory cytokines and 8 measures of glucose homeostasis associated with type 2 diabetes risks. These outcomes were evaluated for their associations with direct measurements of EPA, DHA, and arachidonic acid in RBCs. ANALYSIS: Linear regression was used to detect significant relationships with cytokines and n-3 PUFAs, adiposity, and glucose-related variables. RESULTS: The DHA concentration in RBC membranes was inversely associated with IL-6 (ß = -0.0066; P < 0.001); EPA was inversely associated with TNFα (ß = -0.4925; P < 0.001); and the NIR was inversely associated with Monocyte chemoattractant protein-1 (MCP-1) (ß = -0.8345; P < 0.001) and IL-10 (ß = -1.2868; P < 0.001). CONCLUSIONS: Habitual intake of marine mammals and fish rich in n-3 PUFAs in this study population of Yup'ik Alaska Native adults is associated with reduced systemic inflammation, which may contribute to the low prevalence of diseases in which inflammation plays an important role.

Using Precision Medicine with a Neurodevelopmental Perspective to Study Inflammation and Depression.

PURPOSE OF REVIEW: To consider various precision medicine approaches to further elucidate the relationship between inflammation and depression and to illustrate how a neurodevelopmental perspective can help in this regard. RECENT FINDINGS: Inflammation associates most strongly with phenotypes of depression that reflect illness behavior and/or metabolic dysfunction and obesity. A separate body of research has shown that maternal inflammation during pregnancy can alter brain circuitry important for mood regulation and/or reward in the developing fetus. Our research group is finding that maternal CRP levels differentially predict positive and negative affect in children assessed at age 4 years, depending on the timing of plasma sampling during pregnancy and the sex of the child. Recent authors have stressed the need to use a variety of precision medicine approaches to refine our understanding of inflammation-depression links. Adding a neurodevelopmental perspective may help to address some of the methodological challenges in this active area of study.

Folic acid supplement use and breast cancer risk in BRCA1 and BRCA2 mutation carriers: a case-control study.

PURPOSE: Supplemental folic acid (the more bioavailable and synthetic form of folate) and breast cancer risk in BRCA mutation carriers have not been studied. We evaluated folic acid, vitamin B6 and vitamin B12 supplement use, and breast cancer risk among BRCA mutation carriers. METHODS: In this case-control study, dietary supplement use was collected from BRCA mutation carriers living in Canada. Supplement use was categorized as never or ever use. Total average daily supplement use was categorized as never, moderate, and high use based on tertiles. Unconditional logistic regression was used to estimate the odds ratio (OR) and 95% confidence intervals (CI) for supplement use and breast cancer risk. RESULTS: We included 129 breast cancer cases and 271 controls. Women who used any folic acid-containing supplement had a significantly decreased risk of breast cancer compared to women who never used a folic acid-containing supplement (OR 0.45; 95%CI 0.25, 0.79; P = 0.006). This was significant for BRCA1 mutation carriers only. The OR for moderate folic acid supplement intake was 0.39; P = 0.01, and high intake was 0.54; P = 0.09, compared to never users. Moderate vitamin B12 supplement intake was associated with decreased risk of breast cancer compared to never use (OR 0.48; 95%CI 0.24, 0.96; P = 0.04). CONCLUSIONS: In this first investigation of folic acid supplement use and breast cancer risk in BRCA mutation carriers, these findings suggest that moderate folic acid- and vitamin B12-containing supplement use may be protective for BRCA-associated breast cancer, particularly among BRCA1 mutation carriers. Future studies with larger samples and prospective follow-up are needed.

HLA-F on HLA-Null 721.221 Cells Activates Primary NK Cells Expressing the Activating Killer Ig-like Receptor KIR3DS1.

NK cells elicit important responses against transformed and virally infected cells. Carriage of the gene encoding the activating killer Ig-like receptor KIR3DS1 is associated with slower time to AIDS and protection from HIV infection. Recently, open conformers of the nonclassical MHC class Ib Ag HLA-F were identified as KIR3DS1 ligands. In this study, we investigated whether the interaction of KIR3DS1 on primary NK cells with HLA-F on the HLA-null cell line 721.221 (221) stimulated KIR3DS1+ NK cells. We used a panel of Abs to detect KIR3DS1+CD56dim NK cells that coexpressed the inhibitory NK cell receptors KIR2DL1/L2/L3, 3DL2, NKG2A, and ILT2; the activating NK cell receptors KIR2DS1/S2/S3/S5; and CCL4, IFN-γ, and CD107a functions. We showed that both untreated and acid-pulsed 221 cells induced a similar frequency of KIR3DS1+ cells to secrete CCL4/IFN-γ and express CD107a with a similar intensity. A higher percentage of KIR3DS1+ than KIR3DS1- NK cells responded to 221 cells when either inclusive or exclusive (i.e., coexpressing none of the other inhibitory NK cell receptors and activating NK cell receptors detected by the Ab panel) gating strategies were employed to identify these NK cell populations. Blocking the interaction of HLA-F on 221 cells with KIR3DS1-Fc chimeric protein or anti-HLA-F Abs on exclusively gated KIR3DS1+ cells reduced the frequency of functional cells compared with that of unblocked conditions for stimulated KIR3DS1+ NK cells. Thus, ligation of KIR3DS1 activates primary NK cells for several antiviral functions.

Oxidized Low-Density Lipoprotein Loading of Macrophages Downregulates TLR-Induced Proinflammatory Responses in a Gene-Specific and Temporal Manner through Transcriptional Control.

Hypercholesterolemia is a key risk factor for atherosclerosis and leads to the uptake of native and oxidized low-density lipoprotein (oxLDL) by macrophages (Mϕs) and foam cell formation. Inflammatory processes accompany Mϕ foam cell formation in the artery wall, yet the relationship between Mϕ lipid loading and their response to inflammatory stimuli remains elusive. We investigated proinflammatory gene expression in thioglycollate-elicited peritoneal Mϕs, bone marrow-derived Mϕs and dendritic cells, and RAW264.7 cells. Loading with oxLDL did not induce peritoneal Mϕ apoptosis or modulate basal-level expression of proinflammatory genes. Upon stimulation of TLR4, the rapid induction of IFN-ß was inhibited in cells loaded with oxLDL, whereas the induction of other proinflammatory genes by TLR4 (LPS), TLR3 (polyriboinosinic-polyribocytidylic acid), TLR2 (Pam3CSK4), and TLR9 (CpG) remained comparable within the first 2 h. Subsequently, the expression of a subset of proinflammatory genes (e.g., IL-1ß, IL-6, CCL5) was reduced in oxLDL-loaded cells at the level of transcription. This phenomenon was partially dependent on NF erythroid 2-related factor 2 (NRF2) but not on nuclear liver X receptors α and ß (LXRα,ß), peroxisome proliferator-activated receptor-γ (PPARγ), and activating transcription factor 3 (ATF3). LPS-induced NF-κB reporter activity and intracellular signaling by NF-κB and MAPK pathways were comparable in oxLDL-loaded Mϕs, yet the binding of p65/RelA (the prototypic NF-κB family member) was reduced at IL-6 and CCL5 promoters. This study revealed that oxLDL loading of Mϕs negatively regulates transcription at late stages of TLR-induced proinflammatory gene expression and implicates epigenetic mechanisms such as histone deacetylase activity.

A Developmental Toxicology Assay Platform for Screening Teratogenic Liability of Pharmaceutical Compounds.

Increasing need for proactive safety optimization of pharmaceutical compounds has led to generation and/or refinement of in vitro developmental toxicology assays. Our laboratory has developed three in vitro developmental toxicology assays to assess teratogenic liability of pharmaceutical compounds. These assays included a mouse molecular embryonic stem cell assay (MESCA), a dechorionated zebrafish embryo culture (ZEC) assay, and a streamlined rat whole embryo culture (rWEC) assay. Individually, the assays presented good (73-82%) predictivity. However, it remains to be determined whether combining or tiering the assays could enhance performance. Seventy-three compounds representing a broad spectrum of pharmaceutical targets and chemistry were evaluated across the assays to generate testing strategies that optimized performance. The MESCA and ZEC assays were found to have two limitations: compound solubility and frequent misclassification of compounds with H1 receptor or GABAnergic activity. The streamlined rWEC assay was found to be a cost-effective stand-alone assay for supporting poorly soluble compounds and/or ones with H1 or GABAnergic activity. For all other compounds, a tiering strategy using the MESCA and ZEC assays additionally optimized throughput, cost, and minimized animal use. The tiered strategy resulted in improved performance achieving 88% overall predictivity and was comparable with 89% overall predictivity achieved with frequency analysis (final teratogenic classification made from most frequent teratogenic classification from each individual assay). Furthermore there were 21 compounds in the test set characterized as definitive or suspect human teratogens and the multiassay approach achieved 95 and 91% correct classification using the tiered or frequency screening approach, respectively.

Optimization and Performance Assessment of the Chorion-Off [Dechorinated] Zebrafish Developmental Toxicity Assay.

The Dechorinated Zebrafish Embryo Developmental toxicity assay was originally developed from a training set of 31 compounds and reported to be 87% concordant with in vivo teratogenicity data (Brannen, K. C., Panzica-Kelly, J. M., Danberry, T. L., and Augustine-Rauch, K. A. (2010). Development of a zebrafish embryo teratogenicity assay and quantitative prediction model. Birth Defects Res. 89, 66-77.). The assay includes scoring larva treated in a concentration range for malformations of specific morphological structures/organ systems. The model includes identifying a no-adverse-effect-level (NOAEL) and the concentration resulting in 25% lethality (LC25) at 5 days postfertilization. An LC25/NOAEL ratio ≥10 classifies a compound positive for teratogenic potential. A consortium effort evaluated a modified version of this assay which involved enzymatic chorion treatment instead of manual dissection and used experimental replicates for final classification. The modified assay achieved an 85% overall predictivity (Gustafson, A. L., Stedman, D. B., Ball, J., Hillegass, J. M., Flood, A., Zhang, C. X., Panzica-Kelly, J., Cao, J., Coburn, A., Enright, B. P., et al. (2012). Inter-laboratory assessment of a harmonized zebrafish developmental toxicology assay - progress report on phase I. Reprod. Toxicol. 33, 155-164.). The objective of this study was to perform a thorough performance evaluation of the dechorinated assay by repeating the original training set and testing additional compounds in experimental replicates. When the initial training set was repeated with inclusion of experimental replicates, the overall predictivity was 83%. Model performance was tested with an additional 34 compounds and achieved overall predictivity of 74%. When the training and test sets were combined (63 compounds) the assay's final sensitivity was 83% and the specificity was 71%. Total predictivity was 78% with relatively balanced predictivity for nonteratogens (77%) and teratogens (78%). The chorion-off assay achieved superior sensitivity (83%) compared with sensitivity (63-74%) reported by consortium efforts testing a similar assay with chorion-intact embryos (Ball, J. S., Stedman, D. B., Hillegass, J. M., Zhang, C. X., Panzica-Kelly, J., Coburn, A., Enright, B. P., Tornesi, B., Amouzadeh, H. R., Hetheridge, M., et al. (2014). Fishing for teratogens: a consortium effort for a harmonized zebrafish developmental toxicology assay. Toxicol. Sci. 139, 210-219.). Additional protocol modifications were made to increase assay throughput.

Fishing for teratogens: a consortium effort for a harmonized zebrafish developmental toxicology assay.

A consortium of biopharmaceutical companies previously developed an optimized Zebrafish developmental toxicity assay (ZEDTA) where chorionated embryos were exposed to non-proprietary test compounds from 5 to 6 h post fertilization and assessed for morphological integrity at 5 days post fertilization. With the original 20 test compounds, this achieved an overall predictive value for teratogenicity of 88% of mammalian in vivo outcome [Gustafson, A. L., Stedman, D. B., Ball, J., Hillegass, J. M., Flood, A., Zhang, C. X., Panzica-Kelly, J., Cao, J., Coburn, A., Enright, B. P., et al. (2012). Interlaboratory assessment of a harmonized Zebrafish developmental toxicology assay-Progress report on phase I. Reprod. Toxicol. 33, 155-164]. In the second phase of this project, 38 proprietary pharmaceutical compounds from four consortium members were evaluated in two laboratories using the optimized method using either pond-derived or cultivated-strain wild-type Zebrafish embryos at concentrations up to 100µM. Embryo uptake of all compounds was assessed using liquid chromatography-tandem mass spectrometry. Twenty eight of 38 compounds had a confirmed embryo uptake of >5%, and with these compounds the ZEDTA achieved an overall predictive value of 82% and 65% at the two respective laboratories. When low-uptake compounds (≤ 5%) were retested with logarithmic concentrations up to 1000µM, the overall predictivity across all 38 compounds was 79% and 62% respectively, with the first laboratory achieving 74% sensitivity (teratogen detection) and 82% specificity (non-teratogen detection) and the second laboratory achieving 63% sensitivity (teratogen detection) and 62% specificity (non-teratogen detection). Subsequent data analyses showed that technical differences rather than strain differences were the primary contributor to interlaboratory differences in predictivity. Based on these results, the ZEDTA harmonized methodology is currently being used for compound assessment at lead optimization stage of development by 4/5 of the consortium companies.

Establishment of a molecular embryonic stem cell developmental toxicity assay.

The mouse embryonic stem cell test (EST) is a 10-day screen for teratogenic potential developed to reduce animal use for embryotoxicity testing of chemicals (Spielmann, 2005; Spielmann et al., 1997). In this study, we used the cytotoxicity IC(50) values and transcriptional expression changes as primary endpoints in a shorter 4-day version of the EST, the molecular embryonic stem cell assay. Mouse D3 embryonic stem cells were used for cytotoxicity assessment (monolayers) or grown as embryoid bodies in low attachment plates for transcriptional profiling. Sixty-five compounds with known in vivo teratogenicity (33 teratogens and 32 nonteratogens) were evaluated to develop a model for classifying compounds with teratogenic potential. The expression of 12 developmentally regulated gene targets (nanog, fgf5, gsc, cd34, axin2, apln, chst7, lhx1, fgf8, sox17, foxa2, and cxcr4) was measured following exposure of embryoid bodies to a single compound concentration (0.1 × the cytotoxicity IC(20)) for 4 days. In the decision-tree model, compounds with IC(50) values < 22 µM were categorized as teratogens, whereas compounds in the two groups with IC(50) values between 22-200 µM and > 200 µM were categorized as teratogens if ≥ 8 and 12 genes, respectively, were deregulated by at least 10%. Forty-seven of 65 compounds of the training set were correctly identified (72% total concordance). In a test set of 12 additional compounds (5 teratogens, 7 nonteratogens), 10 were correctly classified by this approach (83% concordance). The false positive rate in the training and test sets was 24 and 0%, respectively, indicating that this assay has potential to identify teratogens.

Zebrafish embryo developmental toxicology assay.

A promising in vitro zebrafish developmental toxicology assay was generated to test compounds for their teratogenic potential. The assay's predictivity is approximately 87% in AB strain fish (Brannen KC et al., Birth Defects Res B Dev Reprod Toxicol 89:66-77, 2010). The procedure entails exposing dechorionated gastrulation-stage embryos to a range of compound concentrations for 5 days throughout embryonic and larva development. The larvae are evaluated for viability in order to identify an LC25 (the compound concentration in which 25% lethality is observed) and morphological anomalies using a numerical score system to identify the NOAEL (no observed adverse effect level). These values are used to calculate the teratogenic index (LC25/NOAEL ratio) of each compound. If the teratogenic index is equal to or greater than 10 then the compound is classified as a teratogen, and if the ratio is less than 10 then the compound is classified as a nonteratogen (Brannen KC et al., Birth Defects Res B Dev Reprod Toxicol 89:66-77, 2010).

A dysmorphology score system for assessing embryo abnormalities in rat whole embryo culture.

BACKGROUND: The rodent whole embryo culture (WEC) system is a well-established model for characterizing developmental toxicity of test compounds and conducting mechanistic studies. Laboratories have taken various approaches in describing type and severity of developmental findings of organogenesis-stage rodent embryos, but the Brown and Fabro morphological score system is commonly used as a quantitative approach. The associated score criteria is based upon developmental stage and growth parameters, where a series of embryonic structures are assessed and assigned respective scores relative to their gestational stage, with a Total Morphological Score (TMS) assigned to the embryo. This score system is beneficial because it assesses a series of stage-specific anatomical landmarks, facilitating harmonized evaluation across laboratories. Although the TMS provides a quantitative approach to assess growth and determine developmental delay, it is limited to its ability to identify and/or delineate subtle or structure-specific abnormalities. Because of this, the TMS may not be sufficiently sensitive for identifying compounds that induce structure or organ-selective effects. METHOD: This study describes a distinct morphological score system called the "Dysmorphology Score System (DMS system)" that has been developed for assessing gestation day 11 (approximately 20-26 somite stage) rat embryos using numerical scores to differentiate normal from abnormal morphology and define the respective severity of dysmorphology of specific embryonic structures and organ systems. This method can also be used in scoring mouse embryos of the equivalent developmental stage. RESULT AND CONCLUSION: The DMS system enhances capabilities to rank-order compounds based upon teratogenic potency, conduct structure- relationships of chemicals, and develop statistical prediction models to support abbreviated developmental toxicity screens.

Morphological score assignment guidelines for the dechorionated zebrafish teratogenicity assay.

BACKGROUND: Recently we reported the development and optimization of a zebrafish teratogenicity assay using dechorionated AB strain embryos, a promising assay that was 87% concordant in correctly identifying in vivo teratogens and non-teratogens from a set of 31 compounds (Brannen et al., 2010: Birth Defects Res 89:66-77). METHODS: This assay utilizes a zebrafish morphological score system to characterize adverse effects and identify the no-observed-adverse-effect level (NOAEL). RESULTS: This report describes in detail the morphological score system used in the dechorionated zebrafish embryo culture teratogenicity assay. The morphological assessment includes evaluation of most structures and organ systems and grades relative severity of abnormalities. CONCLUSIONS: To this end, the morphological score system provides information of tissue-specific teratogenicity that has been found to have good concordance with structures found affected in vivo and can also be used to rank compounds based on the severity of malformations.