Assessment of phthalates/phthalate alternatives in children's toys and childcare articles: Review of the report including conclusions and recommendation of the Chronic Hazard Advisory Panel of the Consumer Product Safety Commission.

The Consumer Product Safety Commission (CPSC) convened a Chronic Hazard Advisory Panel (CHAP) on Phthalates found in children's toys, and childcare products, and in products used by women of childbearing age. The CHAP conducted a risk assessment on phthalates and phthalate substitutes, and made recommendations to either ban, impose an interim ban, or allow the continued use of phthalates and phthalate substitutes in the above products. After a review of the literature, the evaluation included toxic end points of primary concern, biomonitoring results, extant exposure reconstruction, and epidemiological results.

A BAYESIAN GRAPHICAL MODELING APPROACH TO MICRORNA REGULATORY NETWORK INFERENCE.

It has been estimated that about 30% of the genes in the human genome are regulated by microRNAs (miRNAs). These are short RNA sequences that can down-regulate the levels of mRNAs or proteins in animals and plants. Genes regulated by miRNAs are called targets.

Inducible 70 kDa heat shock proteins protect embryos from teratogen-induced exencephaly: Analysis using Hspa1a/a1b knockout mice.

Background: It is well known that a variety of teratogens induce neural tube defects in animals; however, less is known about proteins that play a role in protecting embryos from teratogen-induced neural tube defects. Previously, our laboratory has shown that embryos overexpressing the 70-Da heat shock proteins (HSPs) Hspa1a and Hspa1b were partially protected from the deleterious effects of exposure to hyperthermia in vitro.

Methods: In the present studies, we have used a transgenic mouse in which both of the stress-inducible HSPs Hspa1a and Hspa1b were deleted by homologous recombination.

Gene and microRNA expression in p53-deficient day 8.5 mouse embryos.

Background: Neural tube defects (NTDs) are one of the most common human birth defects, with a prevalence of approximately 1 in 1000 live births in the United States. In animal studies, deletion of p53 leads to a significant increase in embryos that exhibit exencephaly. Whereas several studies have closely investigated the morphologic changes of p53-deficient embryos, no study has reported the molecular-level alteration in p53-deficient embryos.

Effects of maternal hyperthermia on myogenesis-related factors in developing upper limb.

Background: Maternal hyperthermia is one causative factor in various congenital anomalies in experimental animals and humans. In the present study, we assessed the effects of high temperature on limb myogenesis in mice.

Methods: Pregnant mice, C57BL/6 strain, were exposed to hyperthermia (43 degrees C, 5 minutes) on embryonic day (ED) 8.

The roles of p53 and p21 in normal development and hyperthermia-induced malformations.

Background: Hyperthermia (HS) is a well-studied teratogen that induces serious malformations, including neural tube defects. Our previous studies have shown that HS induces apoptosis by activating the mitochondrial apoptotic pathway. Prior to activation of the mitochondrial apoptotic pathway, HS also activates p53 and its target genes.

State of the art in developmental toxicity screening methods and a way forward: a meeting report addressing embryonic stem cells, whole embryo culture, and zebrafish.

A meeting was convened so that users of three models for in vitro developmental toxicity (embryonic stem cells, whole embryo culture, and zebrafish) could share their experiences with each model, and explore the areas for improvement. We present a summary of this meeting and the recommendations of the group.

Cell death in normal and abnormal development.

Research over the past 50 years has consistently documented that cell death is an integral part of both normal development and the etiology of birth defects; however, the significance of this cell death has been, until recently, unclear. Research published during the past 15 years has now shown that programmed cell death (PCD) and teratogen-induced cell death are genetically controlled processes (apoptosis) that play important roles in both normal and abnormal development. Therefore, the purpose of this review is to highlight what is known about PCD and teratogen-induced cell death and their relationships to the mechanisms of apoptosis and abnormal development.

Teratogen-induced activation of p53 in early postimplantation mouse embryos.

Hyperthermia (HS) and 4-hydroperoxycyclophosphamide (4CP) activate the mitochondrial apoptotic pathway in day 9 mouse embryos. Previous microarray analyses Microarray analyses revealed that several p53 target genes are upregulated after exposure to HS or 4CP, suggesting a role for p53 in teratogen-induced apoptosis. To explore the role of p53, we assessed the activation of p53 in day 9 mouse embryos exposed to HS or 4CP in vitro.

Proteomics in developmental toxicology.

The objective of this presentation is to review the major proteomic technologies available to developmental toxicologists and, when possible, to provide examples of how various proteomic technologies have been used in developmental toxicology or toxicology in general. The field of proteomics is too broad for us to go into great depth about each technology, so we have attempted to provide brief overviews supplemented with many references that cover the subjects in more detail. Proteomics tools produce a global view of complex biological systems by examining complex protein mixtures using large-scale, high-throughput technologies.

Alterations in gene expression induced in day-9 mouse embryos exposed to hyperthermia (HS) or 4-hydroperoxycyclophosphamide (4CP): analysis using cDNA microarrays.

Teratogen-induced alterations in gene expression play an important role in the genesis of malformations in animals. The recent development of DNA microarrays now offers the opportunity to monitor global changes in gene expression and therefore the potential to obtain significant new information concerning both normal and abnormal development. RNA was isolated from day-9 mouse embryos at 1 and 5 h after exposure to hyperthermia (HS) or 4-hydroperoxycyclophosphamide (4CP) and compared to RNA isolated from concurrent controls using mouse cDNA microarrays.

Alterations in mitochondrial morphology are associated with hyperthermia-induced apoptosis in early postimplantation mouse embryos.

Background: Previously, we showed that teratogens such as hyperthermia activate the mitochondrial apoptotic pathway in day nine mouse embryos. Activation of this pathway involves an initial release of cytochrome c from intermembranous spaces of the mitochondria into the cytoplasm. Cytoplasmic cytochrome c then activates a caspase cascade resulting in the orderly demise of the cell.

Teratogen-induced activation of caspase-6 and caspase-7 in early postimplantation mouse embryos.

Background: Previous work has shown that teratogens such as hyperthermia (HS), 4-hydroperoxycyclophosphamide (4CP), and staurosporine (ST) induce cell death in day 9 mouse embryos by activating the mitochondrial apoptotic pathway. Key to the activation of this pathway is the activation of a caspase cascade involving the cleavage-induced activation of an initiator procaspase, caspase-9, and the downstream effector procaspase, caspase-3. For example, procaspase-3, an inactive proenzyme of 32 kDa is cleaved by activated caspase-9 to generate a large subunit of approximately 17 kDa and a small subunit of approximately 10 kDa.

Teratogen-induced activation of the mitochondrial apoptotic pathway in the yolk sac of day 9 mouse embryos.

Background: Using vital dyes, we have previously shown that while hyperthermia (HS), 4-hydroperoxycyclophosphamide (4CP), and staurosporine (ST) induce cell death within specific tissues (e.g., neuroepithelium) of day 9 mouse embryos, cells of the heart are resistant to the cell death-inducing potential of these teratogens.

Teratogen-induced activation of caspase-9 and the mitochondrial apoptotic pathway in early postimplantation mouse embryos.

Previously we showed that teratogen-induced cell death in mouse embryos is apoptotic in nature, i.e., involves the release of cytochrome c from mitochondria and the subsequent activation of caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), and internucleosomal DNA fragmentation.

2001 Warkany lecture: to die or not to die, the role of apoptosis in normal and abnormal mammalian development.

Cell death is a common and reproducible feature of the development of many mammalian tissues/organs. Two well-known examples of programmed cell death (PCD) are the cell deaths associated with fusion of the neural folds and removal of interdigital mesenchymal cells during digit formation. Like normal development, abnormal development is also associated with increased cell death in tissues/organs that develop abnormally after exposure to a wide variety of teratogens.

Co-localization of active caspase-3 and DNA fragmentation (TUNEL) in normal and hyperthermia-induced abnormal mouse development.

Background: Previous work has shown that caspase-3 activation and DNA fragmentation, two hallmarks of apoptosis, are induced in day 9 mouse embryos exposed to hyperthermia (43 degrees C); however, the methods used to assess caspase-3 activation (Western blot) and DNA fragmentation (gel electrophoresis) did not allow these apoptotic events to be localized to specific cells within the embryo.

Methods: To co-localize active caspase-3 and DNA fragmentation to specific cells, we used paraffin sections of day 13 mouse limb buds, sections of control and hyperthermia-treated day 9 mouse embryos, and sequential immunohistochemical staining for caspase-3 and TUNEL staining for DNA fragmentation. We used a primary rabbit antibody specific for the active, p17 subunit of caspase-3 and a goat anti-rabbit secondary antibody conjugated to Alexa 594 fluorochrome (red fluorescence) to localize active caspase-3.

Teratogen-induced activation of ERK, JNK, and p38 MAP kinases in early postimplantation murine embryos.

Background: Although many teratogens are known to activate apoptotic pathways culminating in abnormal development, little is known about how the embryo transduces a teratogenic exposure into specific responses. Signal reception and transduction are regulated by a number of signal transduction pathways, including the extracellular signal-regulated protein kinases (ERKs), c-Jun N-terminal kinases (JNKs) and the stress-activated protein kinase, p38.

Methods: To analyze the effects of teratogens on MAP kinases, we used whole embryo culture, Western blot analyses, and antibodies recognizing inactive or active MAP kinases, or both.

Cytochrome c release from mitochondria of early postimplantation murine embryos exposed to 4-hydroperoxycyclophosphamide, heat shock, and staurosporine.

Cell death is an early and common event in the pathogenesis associated with the abnormal development induced by a variety of teratogens. Previously, we showed that the cell death induced in day 9 mouse embryos by three teratogens, hyperthermia (HS), 4-hydroperoxycyclophosphamide (4-CP), and sodium arsenite (As), is apoptotic in nature involving the activation of caspase-3, cleavage of poly(ADP-ribose) polymerase (PARP), and DNA fragmentation. We now show that HS, 4-CP, and staurosporine (ST) induce the release of cytochrome c from mitochondria with kinetics suggesting a causal relationship with the activation of caspase-3 and caspase-2.

Teratogen-induced cell death in postimplantation mouse embryos: differential tissue sensitivity and hallmarks of apoptosis.

Teratogen-induced cell death is a common event in the pathogenesis associated with tissues destined to be malformed. Although the importance of this cell death is recognized, little information is available concerning the biochemistry of teratogen-induced cell death. We show that three teratogens, hyperthermia, cyclophosphamide and sodium arsenite induce an increase in cell death in day 9.

Heat shock protein 70 (Hsp70) protects postimplantation murine embryos from the embryolethal effects of hyperthermia.

Previous work has shown that there is a positive correlation between the induction of Hsp70 and its transient nuclear localization and the acquisition and loss of induced thermotolerance in postimplantation rat embryos. To determine whether Hsp70 is sufficient to induce thermotolerance in postimplantation mammalian embryos, we used a transgenic mouse in which the normally strictly inducible Hsp70 is constitutively expressed in the embryo under the control of a beta-actin promoter. Day 8.

Molecular/cellular biology of the heat stress response and its role in agent-induced teratogenesis.

Available data indicate that heat shock proteins act as chaperones under non-stress conditions by assisting in: (1) the folding of newly synthesized proteins, (2) the intracellular translocation of proteins, and (3) the function of other proteins. As we gain additional information concerning cellular physiology, we may find that heat shock proteins play a key role in many additional cellular functions. When cells experience thermal or chemical stress, heat shock proteins take on a new role, conserved from bacteria to humans, of protecting cells from the detrimental effects of stress.

Induction of thermotolerance in early postimplantation rat embryos is associated with increased resistance to hyperthermia-induced apoptosis.

Previously we reported that hyperthermia (43 degrees C) induces cell death in neurulation stage rat embryos as part of the pathogenesis culminating in abnormal growth and development. We now show that hyperthermia-induced cell death occurs by a process termed apoptosis. DNA fragmentation, a hallmark of apoptosis, was noted as early as 2.

Constitutive synthesis of heat shock protein (72 kD) in human peripheral blood mononuclear cells: implications for use as a clinical test of recent thermal stress.

There is no clinical laboratory marker to enhance the diagnosis of recent thermal stress in humans. The 72 kD heat shock protein, HSP 72, which is rapidly synthesized after heat stress could be useful in the diagnosis of illnesses associated with heat stress. In humans HSP, 72 is rapidly synthesized after thermal stress; however, conflicting data suggest it may also undergo low level constitutive synthesis.