The current practice for the assessment of an agent's potential effects on the developing embryo/fetus includes administration of high, maternally toxic doses to pregnant laboratory animals. For most agents evaluated, developmental effects occur concomitant with maternal toxicity, raising the question as to whether or not the developmental effects are secondary to maternal toxicity. This chapter reviews current knowledge on the relationship between specific manifestations of maternal toxicity and their resulting impact on the developing embryo or fetus. Departing from past approaches that have tended to be more phenomenological in nature, the approach taken here is to delve into the physiology of maternal toxicity by characterizing the key stressors presented to the conceptus, and then assessing the conceptus's ability to cope with such changes. The severity of the maternal effect and its timing during pregnancy are discussed as critical determinants of maternally mediated development toxicity. The chapter closes with some guidance and examples for interpreting data relevant to the maternal toxicity–developmental toxicity relationship, and outlines some newer concepts related to selection of doses for testing.

Context Botanicals are widely consumed as dietary supplements and traditional medicines, yet their developmental and reproductive toxicity (DART) potential is often inadequately characterized. Botanicals are chemically complex and variable, complicating traditional in vivo testing and interpretation. There is a need for fit-for-purpose new approach methodologies (NAMs) to evaluate DART hazards of complex mixtures while reducing reliance on mammalian models.Objective This review describes the Botanical Safety Consortium (BSC) DART Working Group strategy to evaluate a battery of NAMs for screening botanical extracts and outlines the botanicals selected as case studies to assess assay suitability for complex mixtures.Methods Available NAMs relevant to DART were identified and reviewed, including human induced pluripotent stem cell assays (e.g., devTox Quick Predict), zebrafish embryos, Caenorhabditis elegans, transcriptomics with Connectivity Map analysis, in vitro pharmacology profiling, and selected in silico tools. Assays were selected to provide complementary functional, mechanistic, and biomarker coverage across biological levels. Eighteen botanicals were chosen based on published in vivo, mechanistic, livestock, or in vitro evidence indicating no concern, uncertain effects, or established DART hazards.Results The selected NAM battery captures diverse mechanisms relevant to DART. The case study botanicals span clear positives (e.g., locoweed, poison hemlock, cottonseed), suspected positives based on limited data (e.g., bitter melon, goldenseal, rue), and low concern examples (e.g., Asian ginseng), enabling evaluation of assay performance for complex mixtures.Conclusions This strategy paper lays the groundwork to assess the suitability of integrated NAMs for screening botanical DART potential and establishes the foundation for subsequent testing and case study evaluation.

Decahydronaphthalene (DHN), an industrial solvent, was evaluated in OECD TG 421 and TG 443 reproductive toxicity studies in Sprague Dawley rats. In the TG 421, oral doses were 0, 100, 300, or 1000 mg DHN/kg/day. In the TG 443, initial doses of 0, 30, 100, or 300 mg/kg/day were increased to 0, 60, 200, and 600 mg/kg/day on test day 30. High dose TG 421 females exhibited estrous cycle disruption; mid and high dose dams had fewer implantations and pups/litter. High dose F0 females in the TG 443 showed signs of stress: lower body weight, disrupted estrous cycling, higher adrenal and lower thymus weights, adrenocortical hypertrophy and thymic atrophy; these effects were less severe at the mid dose. High and mid dose TG 443 F0 females also had fewer implantations and pups per litter and lower litter weight. F1 females in the TG 443 had nominally fewer F2 pups and lower litter weight. The reproductive effects of DHN indicate impaired ovarian function. Mid and high dose F0 dams in the TG 443 showed clear signs of stress, to which the ovary is known to be sensitive. Therefore, DHN's effects are considered secondary to maternal stress, and not relevant to humans.

Plastic microparticles, a form of microparticles commonly referred to as microplastics (MP), have been the focus of increasing interest for understanding potential human and ecological impacts, including the development of health-based benchmark values. This systematic review critically evaluates 24 mammalian studies reporting reproductive and developmental outcomes, a disproportionately focused research area, with a particular focus on methodological rigor and risk of bias. Fit-for-purpose aspects of selection, performance, and attrition bias were integrated into the critical appraisal to better understand the potential bias studies may have across these domains. All studies received a tier III rating based on the National Toxicology Program's Office of Health Assessment and Translation framework, indicating a high risk of bias and insufficient reliability for risk assessment. Key issues identified across the body of evidence include poor exposure characterization, inadequate outcome assessment, lack of validated test guidelines, and failure to account for critical reproductive parameters such as estrous cycle monitoring and sperm analysis standards. Additionally, discrepancies in the particle characterization and homogeneity of the test material limit comparability and reproducibility across studies. This work highlights the current limitations in the body of evidence in terms of internal and construct validity, which preclude any conclusions on MP-related reproductive toxicity, and details a path forward for investigators to consider in future research.

In their Opinion article, Wise et al. (2023, this issue) express concern about the peer review process in Birth Defects Research (BDR) and other journals, and recommend changes to the process for BDR. I talked with one of the authors prior to submission of their article and we discussed whether publishing the Opinion would be helpful to BDR. When the Opinion came in, I sent it to the BDR editors for comment. Most think the Opinion is helpful in heightening awareness of a problem they are all facing. Others pointed out that issues with peer review are nothing new; one sent a commentary from 15 years ago (Stang et al., 2008), in which the authors bemoaned the low proportion of requests to review that are accepted, and offered ideas for incentives including financial compensation, annual awards for active reviewers, and inviting top reviewers to write a commentary to accompany a reviewed manuscript. Finding capable and willing reviewers is growing more difficult for many journals. The acceptance rate for requests to review for BDR was 46.6% in 2018, and down to 40.9% in 2022, a year in which 1444 requests were sent. It is now not uncommon to send 10–15 requests to get two reviewers. This is frustrating for the editors and delays publication of papers. In 2018, time from submission to acceptance was 73 days—in 2022, it was 98 days. Editors often have to broaden their search for reviewers to unfamiliar scientists who are unfamiliar with BDR. While this can bring a healthy diversity of viewpoints, it can also lead to superficial reviews that do not help the editor decide on the paper. Some journals have begun returning submitted articles to the authors if suitable reviewers are not found within an established time frame. Nonmonetary incentives are helpful, especially for junior scientists, and we will begin to implement that immediately. Wise et al. also suggest discounts on the publisher's books as an alternative to monetary incentives. We will look into that. I also like the idea of inviting top reviewers to write commentaries to feature our most interesting articles. The biggest change proposed by Wise et al. is the adoption of the Transparent Review Model for BDR. They provide evidence for its successful use, including by many Wiley journals. Posting or publishing reviews and reviewers names with the article would be optional for both parties. As long as it is optional, I like this idea. My hesitation is that BDR is the journal of The Society for Birth Defects Research and Prevention (BDRP) a relatively small and close knit group, and foregoing anonymity may inhibit reviewers or negatively personalize reviews for authors. Wise et al. invite comments and discussion in response to their Opinion, and I would like to hear what others think about Transparent Review for BDR, as well as other ideas for improving BDR peer review. I will post a blog and an invitation for comment in the BDRP member community as an easy place for members to express their thoughts on peer review. Of course, submission of Opinion articles to BDR is always welcome. In addition to the incentives mentioned above, I am assembling a new Editorial Advisory Board (EAB) to actively work with BDR editors and the BDRP Publications Committee. I encourage nominations, including self-nominations, by email to my contact address. Thank you to those who have already expressed interest. We need a willing and committed group of people to help improve and grow BDR. Importantly, membership on the EAB is not limited to members of BDRP, and we need a diverse EAB to match our diverse scientific interests and our diverse authors. Thank you to Drs. Wise, Harris, Webster and DeSesso for their thoughtful Opinion and their long-time support of BDR and BDRP. Thank you to the editors of BDR for their dedication in the face of a sometimes daunting task. The readership of BDR is growing, as is the number of papers submitted and the impact factor. We are going in the right direction and the peer review process is essential to the continued success of BDR. Please consider this when you are asked to review a paper for our journal.

BACKGROUND:Some per- and poly-fluoroalkyl substances (PFAS) cause neonatal mortality and lower birth weight in rodents. We constructed an Adverse Outcome Pathway (AOP) network for neonatal mortality and lower birth weight in rodents, comprising three putative AOPs. We then assessed strengths of the evidence for the AOPs and applicability to PFAS. Finally, we considered the relevance of this AOP network to human health.METHODS:Literature searches targeted PFAS, peroxisome proliferator-activated receptor (PPAR) agonists, other nuclear receptors, relevant tissues, and developmental targets. We used reviews of established biology and described results of studies with prenatal PFAS exposure that assessed birth weight and neonatal survival. Molecular initiating events (MIEs) and key events (KEs) were proposed and strengths of KE relationships (KERs), applicability to PFAS, and human relevance were assessed.RESULTS:Neonatal mortality has been observed in rodents following gestational exposure to most longer chain PFAS studied, often coincident with lower birth weight. In AOP 1, PPARα activation and PPARγ activation or downregulation are MIEs; placental insufficiency, fetal nutrient restriction, neonatal hepatic glycogen deficit, and hypoglycemia are KEs leading to neonatal mortality and lower birth weight. In AOP 2, constitutive androstane receptor (CAR) and pregnane X receptor (PXR) activation upregulates Phase II metabolism, lowering maternal circulating thyroid hormones. In AOP 3, disrupted pulmonary surfactant function and PPARγ downregulation cause neonatal airway collapse and mortality from respiratory failure.CONCLUSIONS:It is likely that different components of this AOP network will apply to different PFAS, largely determined by which nuclear receptors they activate. The MIEs and KEs in this AOP network can occur in humans, but differences in PPAR structure and function, and the timeline of liver and lung development, suggest that humans may be less susceptible to this AOP network. This putative AOP network elucidates knowledge gaps and research needed to better understand the developmental toxicity of PFAS.

The toxicology of metals is a fascinating field that has engaged many toxicologists around the world in recent decades, as can be appreciated by the depth and breadth of the scientific information presented in this volume. Nowhere is this more evident than among developmental toxicologists and teratologists. Events demonstrating the teratogenicity of methylmercury and the developmental toxicity of lead in humans rank among the most tragic toxic episodes, while recognition of these effects and subsequent efforts to prevent their recurrence rank among our greatest public health triumphs. The realization of the affects of these metals in humans provided impetus to the then-emerging fields of developmental and reproductive toxicology.

With the start of a new year, there are new faces in new places at Birth Defects Research (BDR), the Journal of the Society for Birth Defects Research and Prevention (BDRP). After many years as an Associate and Deputy Editor of BDR, I have assumed the mantle of Editor-in-Chief. I look forward to working with everyone in BDRP to make our journal the foremost place for publishing and reading about research on birth defects, including clinical, epidemiological, and genetic studies in humans, as well as mechanistic and safety assessment studies in animals, in vitro, and in silico. In this age of big data, artificial intelligence, rapidly expanding laboratory and clinical technologies, and increasing focus on health disparities and inequities, there is a growing need for a transdisciplinary journal like BDR. The other editors and I will be working with Dr. Russ Kirby and the BDRP Publications Committee to increase the scientific and social impact of the journal, as well as its usefulness to BDRP members in their professional lives. We will be querying members and other authors and reviewers to help us in the continuing evolution and growth of BDR, so expect to hear from us soon! Diego F. Wyszynski, M.D., M.H.S, Ph.D., has joined BDR as a new Deputy Editor. Dr. Wyszynski brings broad expertise and international experience and presence to BDR. He earned his Ph.D. in Epidemiology from Johns Hopkins School of Hygiene and Public Health, an M.D. from the University of Buenos Aires, Argentina, and postdoctoral training at NIH. He has worked in academia, government, pharmaceutical companies, and consulting. He is the founder and CEO of Pregistry, a global registry of pregnant people and a leader in the development and conduct of observational studies during pregnancy. Pregistry's mission is “healthy pregnancies, healthy babies,” a mission obviously shared with BDRP. Diego is located in London, and his international stature will help BDR reach new audiences and new contributors. Suzanne Gilboa, MHS, Ph.D., is joining BDR as an Associate Editor. Dr. Gilboa earned her M.H.S. degree from Johns Hopkins University, Bloomberg School of Public Health, and her Ph.D. from the University of North Carolina, Chapel Hill. Dr. Gilboa served in the U.S. Peace Corps in Nicaragua as a Health Educator and Community Development Specialist. She is an Epidemiologist and Senior Research Scientist in the National Center on Birth Defects and Developmental Disabilities at the Centers for Disease Control and Prevention, working in maternal and child health surveillance. Suzanne will add expertise in epidemiology and surveillance to BDR. She has been a member of BDRP and the Teratology Society since 2010 and The National Birth Defects Prevention Network since 2014. Many thanks and much appreciation to Dr. Michel Vekemans, who served with distinction as Editor of BDR, Part A and then as Editor-in-Chief of the unified BDR. The journal has made great strides under Michel's leadership, and he leaves big shoes to fill. Thanks to Dr. Philip Lupo, who is rotating off as Associate Editor to take his position as Vice President-Elect of BDRP. Thanks to Kristin Artinger and Tina Chambers, our other Deputy Editors, and Associate Editors Drs. Christopher Bowman, Simon Conway, Sarah Obican, Sonja Rasmussen, and Gary Shaw. Thanks to Mark Paalman, our longtime representative from Wiley, Kristofer Barr of Wiley, who is working with us now, and the staff of our editorial offices. And, as always, thanks to the great support BDR receives from our BDRP management staff. Finally, thanks to all of you, the readers, authors, and reviewers of Birth Defects Research. Let us work together to make it even better. John M. Rogers, Ph.D. Editor-in-Chief.

Adverse Outcome Pathways (AOPs) (frameworks for organizing knowledge about the etiology of an adverse phenotypic outcome) for developmental toxicity are still in their infancy yet represent the culmination of literally centuries of thinking and experimentation. Throughout history people have wondered about the origins of birth defects. It was not until the late nineteenth century that experimental teratology demonstrated that development of embryos could be predictably perturbed by noxious agents, and not until the twentieth century did those experiments include mammalian species. In the mid- to late twentieth century scientists began to design experiments in multiple species to understand the etiology of birth defects. Progress was slow due to limitations in both knowledge of developmental biology and technology. These studies typically took place in individual scientists' laboratories, so lack of collaboration was another impediment. Nevertheless, ideas and experimentation abounded, as in the case of the notorious human teratogen thalidomide, which spurred research producing dozens of hypotheses about mechanisms of teratogenesis and the biology underlying the stark species differences in sensitivity. Developmental biologists began to uncover molecular signaling pathways critical for normal morphogenesis and new approaches to studying them. In the first decade of the twenty-first century, teratologists, developmental biologists, and toxicologists came together first to advocate exploiting new knowledge about evolutionarily conserved signaling pathways to design assays for developmental toxicity done in vitro or in alternative species, and again to advocate for development and application of new approaches including high-throughput batteries of in vitro assays using human materials and robotic instrumentation to speed the accrual of new knowledge. At the same time, rapid advances in computational capabilities spawned in silico models of morphogenesis and powerful cheminformatic approaches. Online databases now provide public access to an ever-expanding volume of data and information, and collaborative tools provide accessible platforms for collective thinking. For AOPs, this includes the AOPwiki (http://aopkb.org/), a public repository for AOPs across the broad field of toxicology that allows for public comments on, and contributions to, AOPs under development. There are at present few AOPs for developmental toxicity in the AOPwiki, but the opportunities are great for this valuable resource. This chapter will briefly recount some historical milestones in teratology and articulate several illustrative examples of research on mechanisms of normal and abnormal development that have served to provide the bricks and mortar from which AOPS for developmental toxicity can be built. AOPs emerging from this knowledge are presented to demonstrate the value of this approach and finally, remaining hurdles to effectively applying the AOP framework to human risk assessments will be discussed.

Plastics surround us. If we move our gaze to what we use in our daily lives, there are plastics in many guises. We rely on them and would be hard pressed to live without them. Our cell phones, ear buds, credit cards, clothes, shoes, car parts, grocery bags, packaging, food containers, glasses, laboratory ware, water bottles, cigarette butts, linings metal cans, pens, medical tubing, laboratory supplies, fishing gear, toys, baby bottles, sippy cups, pacifiers, all are made up of plastics. The manufacture, use, and disposal of plastics release many long-lasting chemicals into the environment that accumulate and negatively affect flora and fauna, as well as humans. As much as we need plastics, we also need to be aware of how they impact the environment, our health, and that of our children and their children. The authors for this special issue have tackled the "trouble with plastics" from several points of view (Figure 1). We will learn many of the ways in which plastics negatively impact our health and induce birth defects, some that may not manifest until later life. These findings should motivate us to consider with some urgency what we can do to minimize or avoid the negative impact of plastics. Dr. Darbre (Darbre, in this issue) provides an overview of the many chemicals lurking in plastics. Bisphenol A and phthalates are added to plastics, leach from them, and disrupt many hormone systems. We breath them in, eat them, and absorb them through our skin. When we are exposed to these insidious endocrine-disrupting chemicals at a vulnerable "window of susceptibility" in utero or during early life, consequences may arise later in life in the form of reproductive difficulties, metabolic disorders, thyroid dysfunction, immune dysregulation, adverse neurobehavioral outcomes, or cancer. Dr. Darbre urges further studies to fill the many gaps in our understanding regarding how these ubiquitous chemicals affect our health and that of generations to come. Dr. Duttaroy and team (Basak et al., in this issue) lay out an extensive review of the compounds related to plastics that can, even at low concentrations, affect the development of animal models and likely that of humans. These include "bisphenols (BPA, BPS, BPF), bis(2-ethylhexyl) phthalate, and dibutyl phthalate (DBP)" and also "polybrominated diphenyl ethers (PBDE) and tetrabromobisphenol A (TBBPA)." Plastics-derived endocrine-disrupting compounds and their alternate "safer" substitutes are showing similar adversity on reproductive development in several studies. Animal studies expose that mechanisms of action of these chemicals that leach from plastics can disturb early embryonic and placental development. Evidence is accumulating that these chemicals act through long-lasting epigenetic effects that may explain transgenerational effects. The U.S. Food and Drug Administration (FDA) banned BPA in baby bottles and sippy cups in 2012. We can congratulate ourselves for reducing our exposure to BPAs, yet its replacements, bisphenol-F (BPF) and bisphenol-S (BPS), may be worse! As the evidence mounts for the negative effects of these chemicals, intriguing questions come to mind. Could the rising levels of these compounds in humans account for the decline in sperm count and the rapid rise in obesity and metabolic disease? Dr. Montor and his team (Segovia-Meurdoza et al., in this issue; Solleiro-Villavicencio et al., in this issue) supply us with the astonishing statistics about the plastic patches covering our planet. These plastic patches or islands are deep, and debris often sinks to the bottom of the ocean. Various animals ingest plastics, and they are ingested in turn up the food chain, eventually reaching large predators including humans. Aside from aesthetic considerations, these patches pose a threat to life in the oceans and to our health in many ways. The two papers from this group also expand on the effects of BPA, phthalates, and other chemicals that are associated with plastics that affect the neuroendocrine (Solleiro-Villavicencio et al., in this issue) and the immune (Segovia-Meurdoza et al., in this issue) systems. While these systems are discussed separately, they overlap in their functions and affect many organs and systems in our bodies. Our "complex regulatory neuroendocrine network" expresses receptors that BPA and phthalates can bind to and initiate disruptive responses and/or inhibit normal responses even at low concentrations. The links between exposure to these chemicals in utero and early life and neurobehavioral and cognitive dysfunction are also disturbing. The authors also warn that, in studies of the immune system, "… many reports do not consider that the immune response must be studied by challenging the immune components, so there is little information about BPA effects on the immune response during disease." This means that immune system deficiencies caused by BPA and phthalates exposure might be missed by studying basal conditions and that BPA and phthalate effects could be worse or more widespread than we suspect. Dr. Posnack and colleagues (Ramadan et al., in this issue) alert us to the inadvertent hospital-based chemical exposures through the use of plastic medical products, which can lead to cardiovascular consequences. Notably, phthalate concentrations accumulate over time in stored red blood cell units with di(2-ethylhexyl)phthalate (DEHP) levels increasing over time. Cardiopulmonary bypass is known to precipitate an inflammatory response, yet it is still unclear whether phthalate and bisphenol chemicals from the tubing circuitry may contribute to these effects. These distressing facts suggest that vulnerable pediatric populations are at increased risk for chemical exposures through lines and other medical devices. Dr. Atay and colleagues (Atay et al., in this issue) describe how BPA affects early chicken embryo development. Key events at these stages are the closure of the neural tube and formation of somites. Both were disrupted by BPA exposure, and the overall growth of the embryo lagged. The puzzle here is that BPA is thought to act as an endocrine disruptor targeting steroid hormone receptors [estrogen receptor (ER), estrogen-related receptor (ERR), and androgen receptor (AR)] (Asencio-Hernández, Kieffer, & Delsuc, 2016) that are known to be expressed during gonad development but have not been reported in the neural tube or somites at this early stage. However, there is a report that these receptors are expressed in the extraembryonic membranes (Kumar, Lohrentz, Gahr, & Groothuis, 2019), suggesting an indirect effect through these membranes rather than a direct effect of BPA on the embryo. Atay et al. report the novel finding that AR and ER mRNAs are expressed on extraembryonic membranes (EMs) before the embryo begins its own hormone production, identifying a novel substrate for action of maternal hormones on the embryo. Finished plastic products that end up in the environment, as well as chemicals released from plastics, are known to negatively affect human and animal health. This special issue provides insight into a number of known and potential health impacts of chemicals in plastics. The ongoing saga of BPA and bisphenol replacements can also be followed in an in-progress special issue of Toxicology, to which articles continue to be added: https://www.sciencedirect.com/journal/toxicology/special-issue/10DGCJPVNJZ. The global problem of plastic manufacture, use, and disposal continues to receive much-needed attention from regulatory bodies and consumer groups worldwide, incorporating new technologies. The authors declare no conflict of interest.

Despite many years and dollars spent on public education to expose the dangers of tobacco smoking and second-hand smoke, smoking persists. Not only that, we now worry about the potential dangers of third-hand smoke (the residue left on surfaces where smoking has occurred), devices offering new ways to smoke (vaping) and the legalization and resultant expansion of the many ways to use cannabis. New research is expanding the vast literature on, and revealing new adverse effects of exposure to tobacco smoke during pregnancy, while nascent research on vaping and cannabis use in pregnancy is just beginning to emerge. Vaping and cannabis are perceived by many to be safe for use in pregnancy, but public health and medical organizations have issued strong warnings against both. This special issue presents focused reviews that lay out what we know about some of the effects of these exposures during pregnancy and the vulnerable adolescent years (Figure 1), and the diverse approaches used to elucidate these effects. We have known for decades that tobacco smoke is a human developmental toxicant and teratogen, and new knowledge continues to emerge about the adverse effects of tobacco smoke on the outcome of pregnancy. We now understand that, in addition to maternal smoking, exposure of pregnant women to second-hand smoke is also developmentally toxic, as is periconceptual paternal smoking. Seven U.S. Surgeon General's Reports since 1964 have addressed the effects of tobacco smoke on pregnancy, the latest of which finds a causal link between maternal smoking and orofacial clefts in offspring and suggestive causal links to clubfoot, gastroschisis and atrial septal defects (USDHHS, 2014). The range of known adverse effects and targets of tobacco smoke exposure during development continues to expand. Surprising links between maternal tobacco smoke exposure during pregnancy and elevated risk of obesity in adolescent offspring (Rogers, in this issue) probably represent the strongest evidence in humans that developmental exposure to environmental chemicals can raise the risk of obesity later in life, and such chemicals have been termed “obesogens” (Janesick & Blumberg, 2016). Nicotine, one of the thousands of chemicals present in tobacco smoke as well as an active ingredient in most electronic cigarettes (also known as Electronic Nicotine Delivery Systems or ENDS) is known to be toxic to the developing brain from studies in both humans and laboratory animals (Hawkey et al., in this issue). Nicotine is also toxic to other developing organs including the lung. Strikingly, studies in rodents suggest that the effects of nicotine can be passed on to future generations even without continued exposure (Kuniyoshi & Rehan, in this issue). The use of ENDS is increasing rapidly with new devices, flavors, and formulations being introduced to the market. Initially appearing around 2007, ENDS have been the most commonly used tobacco-related product among young people in the United States since 2014. Usage increased 78% among high school students between 2017 and 2018. New ENDS products look like USB thumb drives, the most popular brand being JUUL, which always contains nicotine, and which has increased in sales by 600% in 1 year (https://www.cdc.gov/tobacco/basic_information/e-cigarettes/surgeon-general-advisory/index.html). The level of nicotine in electronic cigarette fluids is variable and can be zero or quite high. Fluids are available that have only flavorings and carrier liquid, but recent reports indicate that these can still cause serious health effects. A new study in human volunteers demonstrates that vaping e-liquids, even those that do not contain nicotine, can have acute effects on the vascular endothelium, reducing peak blood flow velocity and luminal flow-mediated dilation in the superficial femoral artery and vein (Caporale et al., 2019). If such changes were to occur in the uteroplacental circulation of pregnant women, dangerous hypoxic conditions could occur for the embryo/fetus. Further, there have been several recent reports of serious lung damage among young ENDS users, prompting an investigation by CDC: https://www.huffpost.com/entry/severe-lung-illness-vaping-nicotine-thc-cdc_n_5d5b6ec4e4b0f667ed679101. The JUUL brand ENDS has among the highest concentrations of nicotine, with one JUUL pod estimated to contain as much nicotine as a pack of tobacco cigarettes. Breland, McCubbin, and Ashford (in this issue) discuss the current products available and patterns of usage, especially among adolescents and perceptions about safety for use in pregnancy. Then, what is currently known about the developmental toxicity of ENDS is reviewed by Greene and Pisano (in this issue). Cannabis legalization continues to expand in the U.S., providing legal and easy access to a growing proportion of the population, including pregnant women. While possession of cannabis is illegal under U.S. federal law, medical use of cannabis is legal in 33 states, four of five U.S. territories and Washington, DC. Recreational use of cannabis is now legal in 11 states and Washington, DC, Guam and the Mariana Islands, and another 15 states have decriminalized use (https://en.wikipedia.org/wiki/Legality_of_cannabis_by_U.S._jurisdiction). The self-reported use of cannabis during pregnancy is estimated at 2–5% in most studies but rises to 15–28% in young, urban, socioeconomically disadvantaged women, and 34–60% of cannabis users continue use during pregnancy (https://www.drugabuse.gov/publications/drugfacts/marijuana). The American College of Obstetrics and Gynecology has issued a committee opinion strongly discouraging cannabis use during pregnancy and by women contemplating pregnancy (ACOG, 2017). Cannabis use during pregnancy is poorly studied and there is a paucity of dose–response data, which is difficult to assess given the increasing and widely variable potency of legal cannabis products. The developing nervous system is a primary target during both the prenatal brain growth spurt and during adolescent maturation. Jacobus, Courtney, and Hodgdon (in this issue) review their work on the effects of cannabis use on the still-developing adolescent human brain as revealed by neuroimaging and neurocognitive assessments. Noncoding RNAs (ncRNAs) are RNAs that do not code for proteins and include microRNAs (miRNAs), long noncoding RNAs (lnRNAs), small interfering RNAs (siRNAs), pseudogenes, and circular RNAs (circRNAs). While ncRNAs do not code for proteins, they can strongly affect the expression of target genes. Expression of ncRNAs has been studied for its potential as a biomarker of diseases including many types of cancer and, during pregnancy, preeclampsia (Wang et al., 2018), and gestational diabetes (Guarino et al., 2018). MicroRNAs are known to regulate gene expression critical to placental development (Hayder, O'Brien, Nadeem, & Peng, 2018). In this issue, this cutting-edge approach is applied to understand the developmental toxicity of drugs of abuse (Pinson & Miranda, in this issue). These authors demonstrate that expression of ncRNAs is sensitive to developmental toxicants including cannabis and nicotine. Social and marketplace forces are driving rapid increases in the use of ENDS and cannabis, and tobacco smoking among pregnant women remains a persistent public health issue. We hope that this special issue of Birth Defects Research helps bring needed increases in research and public policy attention to bear on these widespread exposures. Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Background: After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde-fixable probe that concentrates into acidic compartments of cells and indicates regions of high lysosomal activity and phagocytosis, both of which correlate to apoptotic activity. Thus, LT is a good indicator of apoptosis visualized by confocal microscopy. Results of LT staining of apoptotic cell death correlate well with other whole mount apoptosis vital dyes such as Nile blue sulfate and neutral red, with the added benefit of being fixable in situ. Nile blue sulfate can also be used as a non-vital, nonspecific dye to visualize general morphology. Stains such as acridine orange can be used for surface staining of fixed embryos to yield confocal images that are similar to scanning electron micrographs. Methods: Mouse embryos were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MEOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). Following this treatment, the tissues were nearly transparent. Embryos are mounted on depression slides, and serial sections are imaged by confocal microscopy, followed by 3-D reconstruction. Results: Embryos or tissues as thick as 500 microns (mu m) can be visualized after clearing with BABB. LysoTracker staining reveals apoptotic regions in organogenesis-stage mouse embryos. Morphological observation of tissue was facilitated by combining autofluorescence with Nile blue sulfate staining of fixed embryos or opaque surface staining with acridine orange staining. Conclusions: The use of BABB for clearing LT vital-stained and fixed embryos matches the refractive index of the tissue to the suspending medium, allowing increased penetration of laser light in a confocal microscope. Nile blue sulfate used as a non-vital dye provides a nonspecific staining of fixed embryos that can then be cleared with methyl salicylate for confocal observation. Sample preparation and staining procedures described here, with optimization of confocal laser scanning microscopy, allow for the detection and visualization of morphological structure and apoptosis in embryos up to 500 mu m thick, and stained specimens can be fixed and mounted on depression slides.

Because some adverse health effects associated with chronic arsenic exposure may be mediated by methylated arsenicals, interindividual variation in capacity to convert inorganic arsenic into mono- and di-methylated metabolites may be an important determinant of risk associated with exposure to this metalloid. Hence, identifying biological and behavioral factors that modify an individual’s capacity to methylate inorganic arsenic could provide insights into critical dose-response relations underlying adverse health effects.