From Pipeline to Plant Protection Products: Using New Approach Methodologies (NAMs) in Agrochemical Safety Assessment.

The human population will be approximately 9.7 billion by 2050, and food security has been identified as one of the key issues facing the global population. Agrochemicals are an important tool available to farmers that enable high crop yields and continued access to healthy foods, but the average new agrochemical active ingredient takes more than ten years, 350 million dollars, and 20,000 animals to develop and register. The time, monetary, and animal costs incentivize the use of New Approach Methodologies (NAMs) in early-stage screening to prioritize chemical candidates. This review outlines NAMs that are currently available or can be adapted for use in early-stage screening agrochemical programs. It covers new in vitro screens that are on the horizon in key areas of regulatory concern. Overall, early-stage screening with NAMs enables the prioritization of development for agrochemicals without human and environmental health concerns through a more directed, agile, and iterative development program before animal-based regulatory testing is even considered.

How the Xenopus eleutheroembryonic thyroid assay compares to the amphibian metamorphosis assay for detecting thyroid active chemicals.

The Xenopus Eleutheroembryonic Thyroid Assay (XETA) was recently published as an OECD Test Guideline for detecting chemicals acting on the thyroid axis. However, the OECD validation did not cover all mechanisms that can potentially be detected by the XETA. This study was therefore initiated to investigate and consolidate the applicability domain of the XETA regarding the following mechanisms: thyroid hormone receptor (THR) agonism, sodium-iodide symporter (NIS) inhibition, thyroperoxidase (TPO) inhibition, deiodinase (DIO) inhibition, glucocorticoid receptor (GR) agonism, and uridine 5'-diphospho-glucuronosyltransferase (UDPGT) induction. In total, 22 chemicals identified as thyroid-active or -inactive in Amphibian Metamorphosis Assays (AMAs) were tested using the XETA OECD Test Guideline. The comparison showed that both assays are highly concordant in identifying chemicals with mechanisms of action related to THR agonism, DIO inhibition, and GR agonism. They also consistently identified the UDPGT inducers as thyroid inactive. NIS inhibition, investigated using sodium perchlorate, was not detected in the XETA. TPO inhibition requires further mechanistic investigations as the reference chemicals tested resulted in opposing response directions in the XETA and AMA. This study contributes refining the applicability domain of the XETA, thereby helping to clarify the conditions where it can be used as an ethical alternative to the AMA.

The morphology, functionality, and longevity of a novel all human hepatic cell-based tri-culture system.

There remains a significant need for a convenient, phenotypically stable long-term culture platform for primary human hepatocytes (PHHs) for use in pharmacological and toxicological applications. Conventional in vitro models are often inconvenient, burdensome to use, and unable to support a multitude of donor lots or maintain PHH structural and functional properties over extended time. To address these limitations, an all-human cell-based hepatic tri-culture system (HTCS) has been developed comprised of frozen vials of PHHs and feeder cells. Qualified PHHs exhibited healthy morphological characteristics for ≥30 days. Extensive anastomosing networks of bile canaliculi with tight and gap junctions were established early and remained stable and functional throughout the culture period. After 5 culture days, albumin, urea, and basal Phase 1 and Phase 2 metabolic functions were stable for at least 2 weeks and significantly higher in the HTCS PHHs compared to sandwich monoculture PHHs. Induction of CYP functional activity by prototypical receptor agonists was stable after 4 days for at least 2 weeks. Gene expression of Alb and various CYPs in the HTCS PHHs was significantly higher compared to sandwich monoculture PHHs. The HTCS represents a convenient, phenotypically stable, all-human PHH culture platform for pharmacological and toxicological applications.

Identifying the landscape of developmental toxicity new approach methodologies.

BACKGROUND: The dynamics and complexities of in utero fetal development create significant challenges in transitioning from lab animal-centric developmental toxicity testing methods to assessment strategies based on new approach methodologies (NAMs). Nevertheless, considerable progress is being made, stimulated by increased research investments and scientific advances, such as induced pluripotent stem cell-derived models. To help identify developmental toxicity NAMs for toxicity screening and potential funding through the American Chemistry Council's Long-Range Research Initiative, a systematic literature review was conducted to better understand the current landscape of developmental toxicity NAMs. METHODS: Scoping review tools were used to systematically survey the literature (2010-2021; ~18,000 references identified), results and metadata were then extracted, and a user-friendly interactive dashboard was created. RESULTS: The data visualization dashboard, developed using Tableau® software, is provided as a free, open-access web tool. This dashboard enables straightforward interactive queries and visualizations to identify trends and to distinguish and understand areas or NAMs where research has been most, or least focused. CONCLUSIONS: Herein, we describe the approach and methods used, summarize the benefits and challenges of applying the systematic-review techniques, and highlight the types of questions and answers for which the dashboard can be used to explore the many different facets of developmental toxicity NAMs.

A Transformative Vision for an Omics-Based Regulatory Chemical Testing Paradigm.

Use of molecular data in human and ecological health risk assessments of industrial chemicals and agrochemicals has been anticipated by the scientific community for many years; however, these data are rarely used for risk assessment. Here, a logic framework is proposed to explore the feasibility and future development of transcriptomic methods to refine and replace the current apical endpoint-based regulatory toxicity testing paradigm. Four foundational principles are outlined and discussed that would need to be accepted by stakeholders prior to this transformative vision being realized. Well-supported by current knowledge, the first principle is that transcriptomics is a reliable tool for detecting alterations in gene expression that result from endogenous or exogenous influences on the test organism. The second principle states that alterations in gene expression are indicators of adverse or adaptive biological responses to stressors in an organism. Principle 3 is that transcriptomics can be employed to establish a benchmark dose-based point of departure (POD) from short-term, in vivo studies at a dose level below which a concerted molecular change (CMC) is not expected. Finally, Principle 4 states that the use of a transcriptomic POD (set at the CMC dose level) will support a human health-protective risk assessment. If all four principles are substantiated, this vision is expected to transform aspects of the industrial chemical and agrochemical risk assessment process that are focused on establishing safe exposure levels for mammals across numerous toxicological contexts resulting in a significant reduction in animal use while providing equal or greater protection of human health. Importantly, these principles and approaches are also generally applicable for ecological safety assessment.

A microRNA or messenger RNA point of departure estimates an apical endpoint point of departure in a rat developmental toxicity model.

Traditional developmental toxicity testing practice examines fetal apical endpoints to identify a point of departure (POD) for risk assessment. A potential new testing paradigm involves deriving a POD from a comprehensive analysis of molecular-level change. Here, the rat ketoconazole endocrine-mediated developmental toxicity model was used to test the hypothesis that maternal epigenomic (miRNA) and transcriptomic (mRNA) PODs are similar to fetal apical endpoint PODs. Sprague-Dawley rats were exposed from gestation day (GD) 6-21 to 0, 0.063, 0.2, 0.63, 2, 6.3, 20, or 40 mg/kg/day ketoconazole. Dam systemic, liver, and placenta PODs, along with GD 21 fetal resorption, body weight, and skeletal apical PODs were derived using BMDS software. GD 21 dam liver and placenta miRNA and mRNA PODs were obtained using three methods: a novel individual molecule POD accumulation method, a first mode method, and a gene set method. Dam apical POD values ranged from 2.0 to 38.6 mg/kg/day; the lowest value was for placenta histopathology. Fetal apical POD values were 10.9-20.3 mg/kg/day; the lowest value was for fetal resorption. Dam liver miRNA and mRNA POD values were 0.34-0.69 mg/kg/day, and placenta miRNA and mRNA POD values were 2.53-6.83 mg/kg/day. Epigenomic and transcriptomic POD values were similar across liver and placenta. Deriving a molecular POD from dam liver or placenta was protective of a fetal apical POD. These data support the conclusion that a molecular POD can be used to estimate, or be protective of, a developmental toxicity apical POD.

Bridging Sex-Specific Differences in the CAR-Mediated Hepatocarcinogenesis of Nitrapyrin Using Molecular and Apical Endpoints.

Nitrapyrin, a nitrification inhibitor, produces liver tumors in B6C3F1 mice. In a 2-year oncogenicity study, increased incidence of mice with hepatocellular tumors was observed following exposure to 125 (females only) or 250 mg/kg/day (males and females) nitrapyrin in the diet. Previous data was generated in male mice to support a mode-of-action (MoA) characterized by constitutive androstane receptor (CAR) nuclear receptor (NR) activation, increased hepatocellular proliferation, and subsequent hepatocellular foci and tumor formation. Uncertainty as to the relevance of this MoA for females remained given the increased sensitivity to tumor formation in female mice. A targeted MoA study was conducted to evaluate CAR activation and hepatic responses in female mice treated with the female carcinogenic dose of nitrapyrin for 4 days. Nitrapyrin induced a treatment-related increase in hepatocellular hypertrophy and hepatocellular proliferation. Nitrapyrin also induced a dose-related increase in the Cyp2b10/CAR-associated transcript and liver weights. Nitrapyrin-induced liver weights and Cyp2b10 gene expression for both males and females were compared to data generated from three other established CAR activators; methyl isobutyl ketone, phenobarbital, and sulfoxaflor. The response observed in female mice following exposure to nitrapyrin was within range of the degree of change observed in mice following exposure to tumorigenic doses of other CAR activators. Consistent with the liver MoA in male mice, these data support a CAR-mediated mode of action for nitrapyrin-induced liver tumors in female mice, with the understanding that a focused approach minimizing animal use can bridge male and female datasets when sex-specific carcinogenic differences are observed.

Short-term toxicogenomics as an alternative approach to chronic in vivo studies for derivation of points of departure: A case study in the rat with a triazole fungicide.

The derivation of an apical endpoint point of departure (POD) from animal-intensive testing programs has been the traditional cornerstone of human health risk assessment. Replacement of in vivo chronic studies with novel approaches, such as toxicogenomics, holds promise for future alternative testing paradigms that significantly reduce animal testing. We hypothesized that a toxicogenomic POD following a 14 day exposure in the rat would approximate the most sensitive apical endpoint POD derived from a battery of chronic, carcinogenicity, reproduction and endocrine guideline toxicity studies. To test this hypothesis, we utilized myclobutanil, a triazole fungicide, as a model compound. In the 14 day study, male rats were administered 0 (vehicle), 30, 150, or 400 mg/kg/day myclobutanil via oral gavage. Endpoints evaluated included traditional apical, hormone, and liver and testis transcriptomic (whole genome RNA sequencing) data. From the transcriptomic data, liver and testis biological effect POD (BEPOD) values were derived. Myclobutanil exposure for 14 days resulted in increased liver weight, altered serum hormones, liver histopathology, and differential gene expression in liver and testis. The liver and testis BEPODs from the short-term study were 22.2 and 25.4 mg/kg/day, respectively. These BEPODs were approximately an order of magnitude higher than the most sensitive apical POD identified from the two year cancer bioassay based on testis atrophy (1.4 mg/kg/day). This study demonstrates the promise of using a short-term study BEPOD to derive a POD for human health risk assessment while substantially reducing animal testing.

Integration of novel approaches demonstrates simultaneous metabolic inactivation and CAR-mediated hepatocarcinogenesis of a nitrification inhibitor.

Nitrapyrin, a nitrification inhibitor, produces liver tumors in mice at high doses. Several experiments were performed to investigate molecular, cellular, and apical endpoints to define the key events leading to the tumor formation. These data support a mode-of-action (MoA) characterized by constitutive androstane receptor (CAR) nuclear receptor activation, increased hepatocellular proliferation leading to hepatocellular foci and tumor formation. Specifically, nitrapyrin induced a dose-related increase in the Cyp2b10/CAR-associated transcript and protein. Interestingly, the corresponding enzyme activity (7-pentoxyresorufin-O-dealkylase (PROD) was not enhanced due to nitrapyrin-mediated suicide inhibition of PROD activity. Nitrapyrin exposure elicited a clear dose-responsive increase in hepatocellular proliferation in wild-type mice, but not in CAR knock-out mice, informing that CAR activation is an obligatory key event in this test material-induced hepatocarcinogenesis. Furthermore, nitrapyrin exposure induced a clear, concentration-responsive increase in cell proliferation in mouse, but not human, hepatocytes in vitro. Evaluation of the data from repeat dose and MoA studies by the Bradford Hill criteria and a Human Relevance Framework (HRF) suggested that nitrapyrin-induced mouse liver tumors are not relevant to human health risk assessment because of qualitative differences between these two species.

First-Trimester Urine Concentrations of Phthalate Metabolites and Phenols and Placenta miRNA Expression in a Cohort of U.S. Women.

BACKGROUND: There is increasing concern that early-life exposure to endocrine-disrupting chemicals (EDCs) can influence the risk of disease development. Phthalates and phenols are two classes of suspected EDCs that are used in a variety of everyday consumer products, including plastics, epoxy resins, and cosmetics. In utero exposure to EDCs may affect disease propensity through epigenetic mechanisms. OBJECTIVE: The objective of this study was to determine whether prenatal exposure to multiple EDCs is associated with changes in miRNA expression of human placenta, and whether miRNA alterations are associated with birth outcomes. METHODS: Our study was restricted to a total of 179 women co-enrolled in the Harvard Epigenetic Birth Cohort and the Predictors of Preeclampsia Study. We analyzed associations between first-trimester urine concentrations of 8 phenols and 11 phthalate metabolites and expression of 29 candidate miRNAs in placenta by qRT-PCR. RESULTS: For three miRNAs--miR-142-3p, miR15a-5p, and miR-185--we detected associations between Σphthalates or Σphenols on expression levels (p < 0.05). By assessing gene ontology enrichment, we determined the potential mRNA targets of these microRNAs predicted in silico were associated with several biological pathways, including the regulation of protein serine/threonine kinase activity. Four gene ontology biological processes were enriched among genes significantly correlated with the expression of miRNAs associated with EDC burden. CONCLUSIONS: Overall, these results suggest that prenatal phenol and phthalate exposure is associated with altered miRNA expression in placenta, suggesting a potential mechanism of EDC toxicity in humans.

Epigenome-wide and transcriptome-wide analyses reveal gestational diabetes is associated with alterations in the human leukocyte antigen complex.

BACKGROUND: Gestational diabetes mellitus (GDM) affects approximately 10 % of pregnancies in the United States and increases the risk of adverse health outcomes in the offspring. These adult disease propensities may be set by anatomical and molecular alterations in the placenta associated with GDM. RESULTS: To assess the mechanistic aspects of fetal programming, we measured genome-wide methylation (Infinium HumanMethylation450 BeadChips) and expression (Affymetrix transcriptome microarrays) in placental tissue of 41 GDM cases and 41 matched pregnancies without maternal complications from the Harvard Epigenetic Birth Cohort. Specific transcriptional and epigenetic perturbations associated with GDM status included alterations in the major histocompatibility complex (MHC) region, which were validated in an independent cohort, the Rhode Island Child Health Study. Gene ontology enrichment among gene regulation influenced by GDM revealed an over-representation of immune response pathways among differential expression, reflecting these coordinated changes in the MHC region. This differential methylation and expression may be capturing shifts in cellular composition, reflecting physiological changes in the placenta associated with GDM. CONCLUSIONS: Our study represents the largest investigation of transcriptomic and methylomic differences associated with GDM, providing comprehensive insight into how GDM shapes the intrauterine environment, which may have implications for fetal (re)programming.

The impact of first trimester phthalate and phenol exposure on IGF2/H19 genomic imprinting and birth outcomes.

Genomic imprinting leads to parent-of-origin specific gene expression and is determined by epigenetic modification of genes. The paternally expressed gene insulin-like growth-factor 2 (IGF2) is located about ~100kb from the maternally expressed non-coding gene H19 on human chromosome 11, and both genes play major roles in embryonic and placental growth. Given adverse gestational environments can influence DNA methylation patterns in extra-embryonic tissues, we hypothesized that prenatal exposure to endocrine disrupting chemicals (EDCs) alters H19 and IGF2 methylation in placenta. Our study was restricted to a total of 196 women co-enrolled in the Predictors of Preeclampsia Study and the Harvard Epigenetic Birth Cohort. First trimester urine concentrations of 8 phenols and 11 phthalate metabolites were measured and used to characterize EDC exposure profiles. We assessed methylation of differentially methylated regions (DMRs) by pyrosequencing of H19, IGF2DMR0, and IGF2DMR2 and correlated values with phenol and phthalate metabolites. We also assessed overall expression and allele-specific expression of H19 and IGF2. We found several significant associations between DNA methylation and additive biomarker measurements. A significant decrease in H19 methylation was associated with high levels of the sum (Σ) of phthalate metabolites and metabolites of low molecular weight (LMW) phthalates. Σphthalate and LMW phthalate concentrations were inversely associated with IGF2DMR0 methylation values. Variation in methylation was not associated with changes in allele-specific expression. However increased deviation of allele-specific expression of H19 was associated with Σdi(2-ethylhexyl) phthalate metabolites and high molecular weight phthalates. Neither methylation nor expression of these imprinted regions had a significant impact on birth length or birth weight. Overall, our study provides new insight into an epigenetic mechanism that occurs following EDC exposure.

Anatomical location and redistribution of G protein-coupled estrogen receptor-1 during the estrus cycle in mouse kidney and specific binding to estrogens but not aldosterone.

Prior studies have linked renoprotective effects of estrogens to G-protein-coupled estrogen receptor-1 (GPER-1) and suggest that aldosterone may also activate GPER-1. Here, the role of GPER-1 in murine renal tissue was further evaluated by examining its anatomical distribution, subcellular distribution and steroid binding specificity. Dual immunofluorescent staining using position-specific markers showed that GPER-1 immunoreactivity primarily resides in distal convoluted tubules and the Loop of Henle (stained with Tamm-Horsfall Protein-1). Lower GPER-1 expression was observed in proximal convoluted tubules marked with megalin, and GPER-1 was not detected in collecting ducts. Plasma membrane fractions prepared from whole kidney tissue or HEK293 cells expressing recombinant human GPER-1 (HEK-GPER-1) displayed high-affinity, specific [(3)H]-17ß-estradiol ([(3)H]-E2) binding, but no specific [(3)H]-aldosterone binding. In contrast, cytosolic preparations exhibited specific binding to [(3)H]-aldosterone but not to [(3)H]-E2, consistent with the subcellular distribution of GPER-1 and mineralocorticoid receptor (MR) in these preparations. Aldosterone and MR antagonists, spironolactone and eplerenone, failed to compete for specific [(3)H]-E2 binding to membranes of HEK-GPER-1 cells. Furthermore, aldosterone did not increase [(35)S]-GTP-γS binding to membranes of HEK-GPER-1 cells, indicating that it is not involved in G protein signaling mediated through GPER-1. During the secretory phases of the estrus cycle, GPER-1 is upregulated on cortical epithelia and localized to the basolateral surface during proestrus and redistributed intracellularly during estrus. GPER-1 is down-modulated during luteal phases of the estrus cycle with significantly less receptor on the surface of renal epithelia. Our results demonstrate that GPER-1 is associated with specific estrogen binding and not aldosterone binding and that GPER-1 expression is modulated during the estrus cycle which may suggest a physiological role for GPER-1 in the kidney during reproduction.

Effects of in utero exposure to Bisphenol A or diethylstilbestrol on the adult male reproductive system.

The objective of this study was to determine whether in utero exposure to Bisphenol A (BPA) induced reproductive tract abnormalities in the adult male testis. Using the C57/Bl6 mouse, we examined sex-organ weights, anogenital distance, and testis histopathology in adult males exposed in utero via oral gavage to sesame oil, 50 µg/kg BPA, 1000 µg/kg BPA, or 2 µg/kg diethylstilbestrol (DES) as a positive control from gestational days 10 to 16. No changes in sperm production or germ cell apoptosis were observed in adult testes after exposure to either chemical. Adult mRNA levels of genes associated with sexual maturation and differentiation, GATA4 and ID2, were significantly lower only in DES-exposed testes. In summary, the data indicate no gross alterations in spermatogenesis after in utero exposure to BPA or DES. At the molecular level, in utero exposure to DES, but not BPA, leads to decreased mRNA expression of genes associated with Sertoli cell differentiation.

Akt1 is essential for postnatal mammary gland development, function, and the expression of Btn1a1.

Akt1, a serine-threonine protein kinase member of the PKB/Akt gene family, plays critical roles in the regulation of multiple cellular processes, and has previously been implicated in lactation and breast cancer development. In this study, we utilized Akt1+/+ and Akt1-/- C57/Bl6 female mice to assess the role that Akt1 plays in normal mammary gland postnatal development and function. We examined postnatal morphology at multiple time points, and analyzed gene and protein expression changes that persist into adulthood. Akt1 deficiency resulted in several mammary gland developmental defects, including ductal outgrowth and defective terminal end bud formation. Adult Akt1-/- mammary gland composition remained altered, exhibiting fewer alveolar buds coupled with increased epithelial cell apoptosis. Microarray analysis revealed that Akt1 deficiency altered expression of genes involved in numerous biological processes in the mammary gland, including organismal development, cell death, and tissue morphology. Of particular importance, a significant decrease in expression of Btn1a1, a gene involved in milk lipid secretion, was observed in Akt1-/- mammary glands. Additionally, pseudopregnant Akt1-/- females failed to induce Btn1a1 expression in response to hormonal stimulation compared to their wild-type counterparts. Retroviral-mediated shRNA knockdown of Akt1 and Btn1a1 in MCF-7 human breast epithelial further illustrated the importance of Akt1 in mammary epithelial cell proliferation, as well as in the regulation of Btn1a1 and subsequent expression of ß-casein, a gene that encodes for milk protein. Overall these findings provide mechanistic insight into the role of Akt1 in mammary morphogenesis and function.

The relationship between tumor MSLN methylation and serum mesothelin (SMRP) in mesothelioma.

Malignant pleural mesothelioma (MPM) remains a cancer of poor prognosis. It is hoped that implementation of effective screening biomarkers will lead to earlier diagnoses and improved outcomes. Serum-measured soluble mesothelin-related peptide (SMRP) has been demonstrated to have excellent specificity for MPM, but poor sensitivity precludes its use as a screening biomarker. Using a case series of MPM patients from the International Mesothelioma Program at the Brigham and Women's hospital, we sought to determine whether epigenetic change at the MSLN gene in patient tumors is responsible for the poor sensitivity of SMRP. We identified three potential target regions for CpG methylation silencing in the MSLN promoter, one of which was amenable to bisulfite pyrosequencing and located 214 bp upstream of the transcription start site. MSLN promoter methylation was significantly higher in normal pleura than tumor tissue (P < 6.0x10-9). Next, we compared cases according to serum SMRP status and observed that MSLN methylation was significantly higher among tumors from patients testing negative for SMRP (< 1.5nM) versus those that were SMRP positive (P < 0.03). These results demonstrate that MSLN is normally methylated in the pleura, and that methylation is lost in most tumors. However, in a subset of tumors methylation is retained, and this mechanism explains the poor sensitivity of the SMRP assay. These results may lead to additional biomarker targets that will resolve the poor sensitivity of the SMRP assay and allow implementation of screening among exposed populations.

Subfertility caused by altered follicular development and oocyte growth in female mice lacking PKB alpha/Akt1.

Mammalian females are endowed with a finite number of primordial follicles at birth. Immediately following formation of the primordial follicle pool, cohorts of follicles are either culled from the ovary or are recruited to grow until the primordial follicle population is depleted. The majority of ovarian follicles, including the oocytes, undergo atresia through apoptotic cell death. As PKB alpha/Akt1 is known to regulate apoptosis, we asked whether Akt1 functioned in the regulation of folliculogenesis in the ovary. Akt1(-/-) females display reduced fertility and abnormal estrous cyclicity. At Postnatal Day (PND) 25, Akt1(-/-) ovaries possessed a reduced number of growing antral follicles, significantly larger primary and secondary oocytes, and an increase in the number of degenerate oocytes. By PND90, there was a significant decrease in the number of primordial follicles in Akt1(-/-) ovaries relative to Akt1(+/+). In vivo granulosa cell proliferation was reduced, as were expression levels of Kitl and Bcl2l1, two factors associated with granulosa cell proliferation/survival. No compensation was observed by Akt2 or Akt3 at the mRNA/protein level. Significantly higher serum LH and trends for lower FSH and higher inhibin A and lower inhibin B relative to Akt1(+/+) females were observed in Akt1(-/-) females. Exposure to exogenous gonadotropins resulted in an increase in the number of secondary follicles in Akt1(-/-) ovaries, but few mature follicles. Collectively, our results suggest that PKB alpha/Akt1 plays an instrumental role in the regulation of the growth and maturation of the ovary, and that the loss of PKB alpha/Akt1 results in premature ovarian failure.