Padraig S. J. Malone, MCh, FRCSI, FRCS, FEAPU

• Consultant Paediatric Urologist,
• Southampton University Hospitals, NHS Trust,
• Southampton, United Kingdom

Several regions of human estrogen receptor are involved in the formation of receptor-heat shock protein 90 complexes allergy forecast oahu discount 4mg periactin visa. Cross-talk between the aryl hydrocarbon receptor and hypoxia inducible factor signaling pathways allergy medicine safe during pregnancy purchase 4 mg periactin otc. A thyroid hormone receptor coactivator negatively regulated by the retinoblastoma protein allergy symptoms hay fever symptoms discount periactin 4 mg online. Increase of carcinogenic risk via enhancement of cyclooxygenase-2 expression and hydroxyestradiol accumulation in human lung cells as a result of interaction between BaP and 17-beta estradiol allergy medicine cats buy discount periactin 4mg on line. Ligand-independent regulation of transforming growth factor beta1 expression and cell cycle progression by the aryl hydrocarbon receptor allergy symptoms nose burning order periactin 4 mg with mastercard. Definition of a minimal domain of the dioxin receptor that is associated with Hsp90 and maintains wild type ligand binding affinity and specificity allergy to grass treatment purchase periactin 4 mg on line. Cooperation of heat shock protein 90 and p23 in aryl hydrocarbon receptor signaling. Aryl hydrocarbon receptor imported into the nucleus following ligand binding is rapidly degraded via the cytosplasmic proteasome following nuclear export. Association of the dioxin receptor with the Mr 90,000 heat shock protein: a structural kinship with the glucocorticoid receptor. Serum dioxin-like activity is associated with reproductive parameters in young men from the general Flemish population. Transcriptional activation of c-fos protooncogene by 17beta-estradiol: mechanism of aryl hydrocarbon receptor-mediated inhibition. Maximal aryl hydrocarbon receptor activity depends on an interaction with the retinoblastoma protein. Altered cell cycle control at the G(2)/M phases in aryl hydrocarbon receptor-null embryo fibroblast. Evidence for a novel anti-apoptotic pathway in human keratinocytes involving the aryl hydrocarbon receptor, E2F1, and checkpoint kinase 1. A direct interaction between the aryl hydrocarbon receptor and retinoblastoma protein. Reciprocal regulation of the basic helix-loop-helix/Per-Arnt-Sim partner proteins, Arnt and Arnt2, during neuronal differentiation. Role of heat shock protein 90 dissociation in mediating agonist-induced activation of the aryl hydrocarbon receptor. Simultaneous exposure of rats to dioxin and carbon monoxide reduces the xenobiotic but not the hypoxic response. Endogenous hepatic expression of the hepatitis B virus X-associated protein 2 is adequate for maximal association with aryl hydrocarbon receptor-90-kDa heat shock protein complexes. A truncated Ah receptor blocks the hypoxia and estrogen receptor signaling pathways: a viable approach for breast cancer treatment. The inhibitory mechanisms of the tyrosine kinase inhibitors herbimycin a, genistein, and tyrphostin B48 with regard to the function of the aryl hydrocarbon receptor in Caco-2 cells. Evidence that the co-chaperone p23 regulates ligand responsiveness of the dioxin (Aryl hydrocarbon) receptor. The hsp90 chaperone complex regulates intracellular localization of the dioxin receptor. Mechanism of suppression of cytochrome P-450 1A1 expression by tumor necrosis factor-alpha and lipopolysaccharide. Targeted mutation of the murine arylhydrocarbon receptor nuclear translocator 2 (Arnt2) gene reveals partial redundancy with Arnt. Role of the coiled-coil coactivator (CoCoA) in aryl hydrocarbon receptor-mediated transcription. Functional interaction of nuclear receptor coactivator 4 with aryl hydrocarbon receptor. Novel target genes of the Ah (dioxin) receptor: transcriptional induction of N-myristoyltransferase 2. Distinct roles for aryl hydrocarbon receptor nuclear translocator and ah receptor in estrogen-mediated signaling in human cancer cell lines. Deletion of the aryl hydrocarbon receptor-associated protein 9 leads to cardiac malformation and embryonic lethality. The aryl hydrocarbon receptor displaces p300 from E2F-dependent promoters and represses S phasespecific gene expression. Aromatic hydrocarbon receptor-driven Bax gene expression is required for premature ovarian failure caused by biohazardous environmental chemicals. Aryl hydrocarbon receptor-mediated transcription: ligand-dependent recruitment of estrogen receptor alpha to 2,3,7,8-tetrachlorodibenzo-p-dioxin-responsive promoters. A cellular factor stimulates ligand-dependent release of hsp90 from the basic helix-loop-helix dioxin receptor. Aryl hydrocarbon receptor splice variants in the dioxin-resistant rat: tissue expression and transactivational activity. The intersection between the aryl hydrocarbon receptor (AhR)- and retinoic acid-signaling pathways. A pathway of multi-chaperone interactions common to diverse regulatory proteins: estrogen receptor, Fes tyrosine kinase, heat shock transcription factor Hsf1, and the aryl hydrocarbon receptor. Interactions between aryl hydrocarbon receptor (AhR) and hypoxia signaling pathways. Intrinsic AhR function underlies cross-talk of dioxins with sex hormone signalings. Modulation of oestrogen receptor signalling by association with the activated dioxin receptor. Subcellular localization of the aryl hydrocarbon receptor is modulated by the immunophilin homolog hepatitis B virus X-associated protein 2. Molecular interactions of the aryl hydrocarbon receptor and its biological and toxicological relevance for reproduction. Analysis of aryl hydrocarbon receptor-mediated signaling during physiological hypoxia reveals lack of competition for the aryl hydrocarbon nuclear translocator transcription factor. Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Steroid receptor interactions with heat shock protein and immunophilin chaperones. Two murine homologs of the Drosophila single-minded protein that interact with the mouse aryl hydrocarbon receptor nuclear translocator protein. Activation of transcription factors activator protein-1 and nuclear factor-kappaB by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Aromatic hydrocarbon receptor interaction with the retinoblastoma protein potentiates repression of E2F-dependent transcription and cell cycle arrest. Aldehyde dehydrogenase 3 gene regulation: studies on constitutive and hypoxia-modulated expression. The dioxin receptor controls beta1 integrin activation in fibroblasts through a Cbp-Csk-Src pathway. A novel promoter element containing multiple overlapping xenobiotic and hypoxia response elements mediates induction of cytochrome P4502S1 by both dioxin and hypoxia. Perturbation of the mitosis/apoptosis balance: a fundamental mechanism in toxicology. Trans-activation by the human aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator proteins: direct interactions with basal transcription factors. The transcription factor aryl hydrocarbon receptor nuclear translocator functions as an estrogen receptor beta-selective coactivator, and its recruitment to alternative pathways mediates antiestrogenic effects of dioxin. The silencing mediator of retinoic acid and thyroid hormone receptors can interact with the aryl hydrocarbon (Ah) receptor but fails to repress Ah receptor-dependent gene expression. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A. Unique and overlapping transcriptional roles of arylhydrocarbon receptor nuclear translocator (Arnt) and Arnt2 in xenobiotic and hypoxic responses. Reproductive and developmental effects of occupational pesticide exposure: the epidemiologic evidence. Role of dioxin response element and nuclear factor-kappaB motifs in 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated regulation of Fas and Fas ligand expression. Ligand displaces heat shock protein 90 from overlapping binding sites within the aryl hydrocarbon receptor ligand-binding domain. Ligand promiscuity of aryl hydrocarbon receptor agonists and antagonists revealed by site-directed mutagenesis. Aryl hydrocarbon receptor-deficient mice develop heightened inflammatory responses to cigarette smoke and endotoxin associated with rapid loss of the nuclear factor-kappaB component RelB. Conditional disruption of the aryl hydrocarbon receptor nuclear translocator (Arnt) gene leads to loss of target gene induction by the aryl hydrocarbon receptor and hypoxia-inducible factor 1alpha. Expression of aryl hydrocarbon receptor repressor in normal human tissues and inducibility by polycyclic aromatic hydrocarbons in human tumor-derived cell lines. Involvement of RelB in aryl hydrocarbon receptor-mediated induction of chemokines. Functional and physical interactions between the estrogen receptor Sp1 and nuclear aryl hydrocarbon receptor complexes. Transcriptional activation of cathepsin D gene expression by 17beta-estradiol: mechanism of aryl hydrocarbon receptor-mediated inhibition. Aryl hydrocarbon receptor functions as a potent coactivator of E2F1-dependent trascription activity. Chromium inhibits transcription from polycyclic aromatic hydrocarbon-inducible promoters by blocking the release of histone deacetylase and preventing the binding of p300 to chromatin. Estrogen receptor subtype- and promoter-specific modulation of aryl hydrocarbon receptor-dependent transcription. Aryl hydrocarbon receptor signaling in rainbow trout hepatocytes: role of hsp90 and the proteasome. Cross-talk between basic helix-loop-helix/ per-Arnt-Sim homology transcription factors. Crosstalk between estrogen receptor alpha and the aryl hydrocarbon receptor in breast cancer cells involves unidirectional activation of proteasomes. Influence of liver tumor promoters on apoptosis in rat hepatocytes induced by 2-acetylaminofluorene, ultraviolet light, or transforming growth factor beta 1. Ligand-dependent interactions of the Ah receptor with coactivators in a mammalian two-hybrid assay. Beta tubulin affects the aryl hydrocarbon receptor function via an Arnt-mediated mechanism. The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers. Its activation signifies the ongoing inflammatory processes and is important for combating infections by microorganisms. For example, BaP and related carcinogens are biotransformed and activated by the AhR pathway. RelA, RelB, and c-Rel contain transcriptional activation domains and therefore are transcriptionally active. The p50 and p52 proteins form a transcriptionally active heterodimer with RelA, RelB, or c-Rel. These observations are consistent with reports of the interactions between RelB and AhR in mouse lung tissues (Baglole et al. The AhR is the only member of the family that is known to be activated by xenobiotics, including polyhalogenated aromatic hydrocarbons. The high expression levels of AhR and RelB in lung macrophages are consistent with the results reported from animal studies (Shah et al. The inflammatory responses alter therapeutic indices and increase the toxicity of certain administered drugs. Inflammatory responses also play important roles in pathological conditions of the liver such as drug-induced hepatitis and cholestatic diseases. These interactions have become mechanistic linchpins linking certain pathological responses induced by endogenous and exogenous toxicants (Gu et al. Only a fraction of the cases are known to be caused by highly penetrant familial cancer syndromes such as Lynch syndrome or familial adenomatous polyposis. The majority of cases are likely to be the result of the interaction of low and moderately penetrant genes with environmental/dietary factors. Chronic inflammation has been found to be strongly associated with cancer development. The balance could be disrupted by alterations of the host immune system or pathogenesis of the microbes. These complex molecular interactions warrant further investigation as the intersection of genomics and environmental science continues to be explored. While these ideas are rapidly evolving, it seemed useful to review the emerging concepts as they could inform our understanding of the origins of xenobiotic sensing, as well as the mechanisms that underlie the toxicology of many important environmental contaminants. Therefore, we point to excellent recent reviews that we found helpful in preparing this manuscript (McIntosh et al.

Hypermethylation of CpG island loci of multiple tumor suppressor genes in retinoblastoma allergy symptoms red eyes discount periactin 4mg mastercard. Two ways to fold the genome during the cell cycle: insights obtained with chromosome conformation capture allergy medicine least side effects purchase 4 mg periactin otc. Topological domains in mammalian genomes identified by analysis of chromatin interactions allergy eye pain safe periactin 4mg. Cohesin at active genes: a unifying theme for cohesin and gene expression from model organisms to humans allergy symptoms chest pain purchase line periactin. Hormone-regulated transcriptomes: lessons learned from estrogen signaling pathways in breast cancer cells allergy medicine if allergic to dogs cheap periactin 4 mg mastercard. Selective recognition of acetylated histones by bromodomains in transcriptional co-activators allergy symptoms in july buy periactin 4mg cheap. Regulation of alternative splicing through coupling with transcription and chromatin structure. Myc dynamically and preferentially relocates to a transcription factory occupied by Igh. Transcription factor binding at enhancers: shaping a genomic regulatory landscape in flux. Effect of the anti-androgenic endocrine disruptor vinclozolin on embryonic testis cord formation and postnatal testis development and function. Insulator function and topological domain border strength scale with architectural protein occupancy. Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. Benzo[a]pyrene treatment leads to changes in nuclear protein expression and alternative splicing. Alternative splicing A regulated process during gene expression that results in a single gene coding for multiple proteins. Conformational change A change in shape of a macromolecule, often induced by environmental factors. Dephosphorylation the removal of a phosphate group from a macromolecule or protein. Fourier transformation A method to break a waveform (a function or signal) into an alternate representation, characterized by sine and cosines. Oscillations A dynamic feature of cell signaling pathways that displays persistent and regular variations in magnitude. Phosphorylation the transfer of a phosphate group to a receiving macromolecule or protein. W Qian has been added as co-author and is responsible for updating section 5 "State-of-Art Technologies for Characterizing Protein Phosphorylation". Section 2 "New frontiers in protein kinase regulation" has been updated to discuss the most recent advances in linking oscillatory kinase behaviors to unique gene expression patterns. However, toxicants may deregulate kinase activities by direct and indirect mechanisms. Reductionist approaches have provided an excellent view of toxicant effects on target protein kinases in the context of domain-specific effectsdsetting the stage for integrative approaches to define connectivity between signaling networks. This information is needed to enable the development of predictive frameworks that can be applied to complex biological systems. Finally, new frontiers in protein kinase regulation related to dynamic behaviors signaling networks are discussed that should be considered in toxicological investigations. Eukaryotic organisms possess two general classes of protein kinases, those that transfer phosphate to serine and threonine residues and those transferring phosphate to tyrosine residues. Phosphorylation by protein kinases is recognized as a critical mechanism by which virtually every activity of eukaryotic cells is regulated, including proliferation, gene expression, metabolism, motility, membrane transport, and apoptosis. Reversible phosphorylation can function as a binary switch maintaining proteins in an active or inactive state, depending on the substrate and site of phosphorylation. Many proteins have multiple phosphorylation sites that are regulated by different protein kinases enabling integration of multiple signaling pathways through phosphorylation of a common substrate. Phosphorylation influences many aspects of protein function including activity, tertiary structure, and subcellular distribution. Collectively, these features render the regulation of enzymatic and cellular function by phosphorylation an ideal means of appropriately responding to extra- and intracellular signals. In the pregenomics era, the homologous catalytic-domain amino acid sequences of 65 distinct protein kinases from diverse eukaryotes were aligned (Hanks et al. Construction of the corresponding phylogenetic tree revealed major clusters that included tyrosine-, cyclic nucleotide-, calcium-phospholipid-dependent and calmodulin-dependent kinases, which in turn, provided a basis to categorize protein kinases in respect to substrate specificity and mode of regulation. This knowledge was ultimately used to predict the total number of protein kinases present in a variety of genomes, including the human genome (Manning et al. It is estimated that the human genome encodes genes for 518 different protein kinases that constitute about 1. Further, 13 atypical protein kinase families have been identified that contain proteins reported to have kinase activity, but which lack sequence similarity to the eukaryotic protein kinase domain (examples included the phosphatidylinositol 30 -kinase family). Although sequence similarity is lacking, the atypical protein kinases exhibit structural similarity to the eukaryotic protein kinase domain (Yamaguchi et al. The selective phosphorylation of phosphorylase at Ser14 by phosphorylase kinase illustrates this specificity (Krebs and Fischer, 1964). The 12 conserved subdomains are illustrated by Roman numerals and positions of highly conserved amino acid residues and motifs are indicated above the subdomains (using single-letter amino acid code with X as any amino acid). A detailed discussion of the subdomains and function of conserved residues in relation to crystal structures and catalytic function can be found in Hanks (2003) and references therein. For additional phosphorylation motifs and a review, see Kemp and Pearson (1990) and Ubersax and Ferrell (2007). Phosphate acceptor site is indicated by * and less essential residues are marked X. Studies investigating the sequences surrounding phosphorylation sites have resulted in key advancements regarding consensus phosphorylation motifs (Table 1) that can have subtle variations. It is intriguing that a majority of protein kinases share a common catalytic domain, yet maintain mechanisms to selectively recognize and phosphorylate specific substrates. Differences in the charge and hydrophobicity of surface residues on protein kinases provide an important determinant for achieving this specificity (Ubersax and Ferrell, 2007). Defining phosphorylation motifs is broadly useful to identify candidate proteins that may be regulated by a specific protein kinase. Since site-specific phosphorylation is often predictive of a proteins activity, or infers pathway activation, these motifs are also useful for developing reagents, such as phosphorylation sitespecific antibodies, that enable detailed interrogation of these regulatory features in complex biological systems. In many cases, phosphorylation induces a conformational change in the substrate that stimulates its enzymatic activity, such as in the case of phosphorylase b undergoing a cooperative allosteric transition to phosphorylase a following serine phosphorylation (Barford and Johnson, 1989). Tyrosine kinases often become autophosphorylated within the activation segment of their kinase domains and this event induces conversion to a more active state (Hubbard, 1997; Pawson, 2004). In addition, tyrosine phosphorylation creates specific binding sites for signal transducing proteins, such as those associated with the Src homology domains (Sadowski et al. In fact, families of interaction domains have been identified that selectively recognize sites phosphorylated by serine/threonine and tyrosine kinases (Schlessinger and Lemmon, 2003; Yaffe and Elia, 2001). It is noteworthy that coordination of signaling pathways via interaction domains extends to diverse posttranslational modifications, including acetylation, methylation or ubiquitination of lysine residues, arginine methylation, and proline hydroxylation (Seet et al. Therefore, protein phosphatases are an important class of enzymes that regulate steady-state phosphorylation. Similar to the protein kinases, protein phosphatases are divided into two main families based on their ability to dephosphorylate serine/threonine or tyrosine residues. Although a detailed discussion of phosphatases is beyond the scope of this chapter, this class of enzymes is equally important in regulating cell function and should be carefully considered in the interpretation of biological processes regulated by phosphorylation. As interest in the biological function of protein kinase dynamics advances, new insights into the importance of phosphatase activities in signal transduction are emerging. Specifically, in New Frontiers in Protein Kinase Regulation section we discuss new frontiers in protein kinase regulation associated with dynamic behaviors. In this model, growth factor stimulation induces oscillatory behavior, while oscillations are not observed in the absence of growth factor. This observation raises the possibility that toxicants could induce kinase activation indirectly through inhibiting negative feedback loops and few have considered this mode of action in pathophysiology. The majority of kinase inhibitors show significant cross-reactivity, which is generally an undesirable property. However, in some cases cross-reactivity may contribute to potential practical therapeutic applications. As one example, Imatinib is active against multiple kinases and this property has generated interest in its application for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors (Carella and Lerma, 2007; Gupta et al. Shifting the balance between these pathways has important cell fate implications from both toxicological and therapeutic perspectives. The simultaneous inhibition of two different cyclindependent kinases in Saccharomyces cerevisiae (Cdk1 and Pho85) elicits cellular responses that are not induced by blocking either kinase alone (Kung et al. Therefore, the regulation of multiple kinase activities can have synergistic effects that would not be apparent from the actions ascribed to an individual kinase. Collectively, these observations are consistent with the view that cellular behavior is controlled through a complex orchestration of signaling networks with emergent properties. Finally, the generation of an oxidative stress is associated with many chemical toxicants and the ensuing stress response is associated with the activation of multiple stress-responsive kinases (Matsumura, 2003). Collectively, toxic responses are Protein Kinases 269 often associated with complex kinase regulatory patterns. The availability of convenient pharmacological kinase inhibitors in combination with measurements of target kinase activity has enabled a rapid characterization of individual kinases that contribute to toxic responses. In contrast, far less is known about the interplay between multiple protein kinases that lead to emergent properties influencing toxicity. This information would be useful to develop appropriate screening platforms that could support the intensive efforts being directed at the development of kinase-selective inhibitors; and would have logical implications for comparing mechanisms-of-action for toxicants at environmentally relevant exposure levels relative to higher concentrations often used to interrogate biological systems in an effort to improve signal-to-noise. The function of oscillations is under investigation by several laboratories and is believed to represent a regulatory layer encoding biological information. We also used a curve-fitting approach to characterize the oscillatory waveforms and additional features. Oscillations are a relatively new area of investigation and there is little information available to help interpret how specific deregulation of kinase dynamics may impact pathophysiology. Direct measurements of kinase oscillations in an organism are difficult, if not impractical; therefore, accurate and reliable indices of kinase dynamics that can be extended to experimental animal models are needed (discussed below). Morphogens governing developmental biology clearly illustrate the importance of gradients as a regulatory mode in biology (Baumann, 2014; Plouhinec et al. Molecular gradients associated with pathways exhibiting oscillatory behavior have also been documented (Stamataki et al. In a toxicological context, there are emerging examples of relevant perturbations that could plausibly link a pathophysiological outcome to deregulation of kinase dynamics. Children are uniquely vulnerable to the effects of many chemical, biological and physical agents and the deregulation of kinase oscillations during early developmental periods has not been considered for its possible contributions to pathophysiology. Atomic bomb survivors have shown a significant radiation-associated increase in the risk of cancer associated with adolescent exposure (Preston et al. Thus, there are considerable uncertainties related to toxicant-dependent deregulation of kinase oscillations in developmental biology and pathophysiology. The same challenges in advancing the science apply due to the difficulty with measuring kinase oscillations in an organism and a current research need is to develop reliable molecular indices of kinase oscillations that can bridge in vitro/in vivo model systems. As we begin looking forward, there are hypothetical alternative mechanisms by which toxicants may impact kinase dynamics that warrant discussion. Thus, kinase dynamics can be modulated through targeted disruption of either positive or negative feedback loops. The biological significance of inappropriate activation of kinase oscillations by toxicants has not been investigated. Protein Kinases 271 Finally, exposure to environmental toxicants often occurs at low doses, thus making efforts to define mechanisms of action a challenge due to poor signal-to-noise. A common approach is to use robust exposure conditions where cellular/molecular responses can be conveniently measured and then extrapolate the observable responses to low doses. However, these conventional measures are not adequate to confidently detect changes in kinase oscillations and may miss kinase dynamics occuring at low dose exposures. In fact, it is nearly impossible to imagine a toxicant that would not, at least indirectly, modulate some aspect of protein kinase-dependent signaling. In some cases, the toxicant may directly bind to a kinase and modulate activity. These direct phosphorylation events have been characterized in detail and site-specific phosphorylation patterns have been defined. Less is known of how dynamic kinase behaviors uniquely couple to transcriptional regulation, but plausible examples are beginning to emerge. The latter property quickly caught the attention of researchers in the field of chemical carcinogenesis and growth regulation and initiated an intensive effort to characterize the role of this enzyme in normal growth and differentiation processes. The direct binding of metals to target protein kinases and associated regulation of kinase activity is of broad toxicological interest. As one additional example, c-Src is activated by mercuric chloride in a cysteine-dependent fashion (Senga et al.

Early in the apoptotic process allergy medicine ok while breastfeeding generic periactin 4 mg on line, even when the plasma membrane integrity is relatively well maintained allergy symptoms from eggs buy 4mg periactin with visa, apoptotic cells may lose their plasma membrane potential and activate membrane phospholipid scramblase systems allergy forecast venice italy quality 4 mg periactin. The external plasma membrane leaflet and cytosol of apoptotic cells can be acidified as well allergy symptoms plugged ears order discount periactin line. These alterations differentiate apoptotic cells from viable and necrotic cells (Cohen et al allergy symptoms of pollen discount 4 mg periactin mastercard. The mechanisms underlying this neuroprotection seem to involve improvement of mitochondrial function sulphate allergy symptoms uk buy 4mg periactin otc, antioxidant activity, stabilization of mitochondrial membranes, and/or modulation of protein and gene expression (Scafidi et al. Because of the similarity of the physiology, pharmacology, metabolism, reproductive systems and developmental trajectory and time course of the nonhuman primate to that of the human, they are exceptionally good animal models of neurodevelopment and for the detection of potential neurodegenerative effects associated with exposure to general anesthetics. Such microglial activation usually begins within several hours after toxic insult and lasts for several days (Banati, 2003; Ito et al. The severity of sevoflurane-induced neuronal apoptosis was dependent upon exposure duration with the longer exposures being associated with higher levels of apoptosis (Liu et al. Neural progenitor cell proliferation is critical for normal brain development and for mounting responses to injury. These investigations demonstrated that prolonged exposure to sevoflurane during brain development can cause signification inhibition of neural progenitor cell proliferation that lasts for at least two weeks. These observations suggest increased microglial activation and astrocytosis in response to sevoflurane-induced neuronal effects. Sevoflurane-induced neural effects in the nonhuman primate were observed in multiple brain areas including the frontal cortex and temporal lobe and the thalamus whereas in the rat it was seen primarily in the frontal cortex. These data indicate that the adverse effects associated with general anesthesia (in this case induced by sevoflurane) are expressed in a time- and region-specific fashion (Zhang et al. These approaches will provide important insights into the pathological processes that occur during the expression of neurotoxicity and provide critical details on the time course (life cycle) of neurotoxic events and assist in the identification of new molecular-targets for the development of new therapeutic strategies. Neuropathological imaging: In vivo detection of glial activation as a measure of disease and adaptive change in the brain. Peripheral type benzodiazepine binding sites are a sensitive indirect index of neuronal damage. Brain and behavior changes in 12-month-old Tg2576 and nontransgenic mice exposed to anesthetics. Synthesis and evaluation in monkey of two sensitive 11C-labeled aryloxyanilide ligands for imaging brain peripheral benzodiazepine receptors in vivo. Acetylcarnitine induces heme oxygenase in rat astrocytes and protects against oxidative stress: Involvement of the transcription factor Nrf2. Acetylcarnitine and cellular stress response: Roles in nutritional redox homeostasis and regulation of longevity genes. Biomarker research in neurotoxicology: the role of mechanistic studies to bridge the gap between the laboratory and epidemiological investigations. Reversal through supplementation of acetyl-L-carnitine and N-tert-butyl-alpha-phenyl-nitrone. Feeding acetyl-L-carnitine and lipoic acid to old rats significantly improves metabolic function while decreasing oxidative stress. Anti-apoptotic effect of acetyl-L-carnitine and I-carnitine in primary cultured neurons. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. Neurosensory impairment after surgical closure of patent ductus arteriosus in extremely low birth weight infants: Results from the Trial of Indomethacin Prophylaxis in Preterms. Positron emission tomography imaging of (2R,3R)-5-[(18)F]fluoroethoxybenzovesamicol in rat and monkey brain: A radioligand for the vesicular acetylcholine transporter. Apoptosis-detecting radioligands: Current state of the art and future perspectives. Age-associated mitochondrial oxidative decay: Improvement of carnitine acetyltransferase substrate-binding affinity and activity in brain by feeding old rats acetyl-L-carnitine and/or R-alpha-lipoic acid. Comparison of the effects of L-carnitine and acetyl-L-carnitine on carnitine levels, ambulatory activity, and oxidative stress biomarkers in the brain of old rats. An assessment of the effects of general anesthetics on developing brain structure and neurocognitive function. Anesthetic sevoflurane causes neurotoxicity differently in neonatal naive and Alzheimer disease transgenic mice. Educational attainments in early adolescence of infants who required major neonatal surgery. Presence of peripheral-type benzodiazepine binding sites on human erythrocyte membranes. Peripheral-type benzodiazepine receptor in neurosteroid biosynthesis, neuropathology and neurological disorders. Translocator protein (18 kDa): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function. Ketamine anesthesia during the first week of life can cause long-lasting cognitive deficits in rhesus monkeys. Noninvasive imaging of endogenous neural stem cell mobilization in vivo using positron emission tomography. Neonatal exposure to sevoflurane induces abnormal social behaviors and deficits in fear conditioning in mice. Neuroprotection by acetyl-L-carnitine after traumatic injury to the immature rat brain. Neonatal exposure to sevoflurane in mice causes deficits in maternal behavior later in adulthood. Up-regulation of peripheral benzodiazepine receptor system in hepatocellular carcinoma. The role of the N-methyl-D-aspartate receptor in ketamine-induced apoptosis in rat forebrain culture. Blockade of N-methyl-D-aspartate receptors by ketamine produces loss of postnatal day 3 monkey frontal cortical neurons in culture. Standardized uptake value atlas: Characterization of physiological 2-deoxy-2-[18F]fluoro-D-glucose uptake in normal tissues. Chronic exposure of gestation rat to sevoflurane impairs offspring brain development. Synthesis, biological evaluation and radiochemical labeling of a dansylhydrazone derivative as a potential imaging agent for apoptosis. Protective effects of acetyl L-carnitine on inhalation anesthetic-induced neuronal damage in the nonhuman primate. In vivo monitoring of sevofluraneinduced adverse effects in neonatal nonhuman primates using small-animal positron emission tomography. Isoflurane anesthesia induced persistent, progressive memory impairment, caused a loss of neural stem cells, and reduced neurogenesis in young, but not adult, rodents. The effects of L-carnitine on the combination of, inhalation anesthetic-induced developmental, neuronal apoptosis in the rat frontal cortex. Prolonged exposure to ketamine increases neurodegeneration in the developing monkey brain. Embryonic stem cells Pluripotent stem cells derived from the inner cell mass of preimplantation embryo blastocyst. N9 Clonal cell line derived from brain cell cultures from mouse embryos immortalized with oncogenic retroviruses. Neurosphere A 3-D culture system composed of free-floating clusters of neural stem cells. Organotypic slice culture Slices of tissue that maintain the cellular architecture and were appropriate circuitry while under culture conditions. They serve as scaffolds along which new neurons can travel from site of origin to final destination. Stem cells Multipotent cells which are able to self-renew ad proliferate without limit to produce progeny cells which terminally differentiate into various cell types. Subgranular zone the region of the dentate gyrus of the hippocampus where granule cell precursors reside. T cells A lymphocyte developed in the thymus that circulates in the blood and lymph and actively participates in the immune response. In vitro cell culture techniques have been successfully used to investigate specific questions of cell biology and nervous system functioning and as such provide a means to systematically study underlying mechanisms related to normal functioning, disease disruption, and pharmacological or toxicological effects. Model systems can be designed to be specific for known cellular mechanisms and to provide a convenient experimental tool for testing possible functions or postulates in vivo. Thus, in vitro models are used for a direct investigation of specific biological processes and to allow for the assessment to be conducted in an isolated context. It is generally acknowledged that in vitro systems provide partial answers to more complex integrated functions and therefore they can supplement but rarely replace investigations within the whole organism. Knowledge gained on underlying biological mechanisms enhances the ability to examine more sophisticated and subtle biologically based expressions of nervous system activity or toxicity and is essential for identifying effects that translate across species. Ideally, in vitro and in vivo studies should complement one another, serving in a back and forth process to identify underlying mechanisms of cellular responses and potential adverse outcomes. Although the assessment of neurotoxic endpoints in the whole animal are presumed causally related to those initiated at the cellular level, with the exception of a few outcomes. While powerful, in vitro systems are limited in assessing delayed responses, identifying effects that are directly related to maturation or specific age of exposure, or discriminating between transient and persistent effect. Speculation of an adverse effect on the functioning organism based on an in vitro observation is inappropriate in the absence of empirical data to support multiple stages in the progression to a functional adverse outcome observed in vivo. However, such mechanistic information is invaluable as a basis for the design of targeted and relevant in vivo studies. Confirmation of such extrapolation can then be used to refine the in vitro models for further detailed examination or the identification of potential compounds with neurotoxic potential. In addition, gaining a better understanding of the underlying mechanisms associated with cell responses and the influence of cell-cell interactions will aid in the determination of whether an effect observed in vitro may also result in an effect in vivo. This article is not intended to review chemical-related findings from in vitro systems or to set a platform for an expanded use of in vitro systems in the screening for neurotoxicity. It is however, intended to present an overview of some of the relevant advantages and limitations of various culture systems, the types of data that can be obtained from such systems, and provide fundamental principles of issues as they relate to assessing neurotoxicity using in vitro systems. The reader is referred to previous detailed reviews on the utility of in vitro models in neurotoxicity assessments (Abdulla and Campbell, 1993; Baumann et al. These range from a loss of trophic factor stimulation, shifts in neurotransmitter signaling, loss of appropriate afferent or efferent connections, shifts in ion channel opening for cell signaling, and more recently influence of systemic factors from the immune system. While these common mechanisms are relatively accepted, translating changes that occur within an in vitro system to the specificity of damage that occurs in the intact nervous system following different types of exposure becomes quite difficult. The in vivo reactions of neurons to injury can vary dramatically as can damage to non-neuronal cells. As examples, neuronal death can be induced by a direction action on the perikaryon, a loss or overstimulation of synaptic target site influence, or deprivation of trophic factors. Rarely do neurotoxicants affect neurons in all regions of the brain but instead they selectively affect neurons, and often do not selectively affect the neuronal populations that are routinely used in culture studies. In addition, often the primary neuropathology observed does not involve neurons but rather non-neuronal sites such as the myelin sheath, the oligodendrocyte, the astrocyte, or microglia. In vitro mechanisms by which non-neuronal cells of the brain are perturbed in a manner that would lead to deficits in brain function are not as clearly defined as those identified for neurons. Each cell type may contribute to the overall manifestation of neurotoxicity not only individually, but as part of a complex cell-cell interactive process that is altered or initiated during the injury response. Often the pattern of pathology reflects the severity and duration of the injury and the acute or chronic nature of exposure. The response of cells can be relative abrupt or a progressive process depending on the underlying mechanism. How to translate findings observed in vitro that often relate to mechanisms of cell death to the specific and unique pathology that is observed with neurotoxicants becomes a challenge. Additionally, classifying a compound as a neurotoxicant makes the implicit assumption In Vitro Systems in Neurotoxicological Studies 453 that any determining effect observed in vitro would either be selective for nervous system cells or would occur at a dose level lower than any toxic effects observed in other cell types. Without such specificity, one is limited in classifying a compound as neurotoxic when the effects are simply associated with general cytotoxicity. However, these challenges, if met, will significantly contribute to our ability to identify underlying mechanisms that can then be appropriately modeled in an in vitro system. Substances can be added or withdrawn from the culture medium, allowing precise temporal analysis of the sequence of events. Depending on the model system, delivery of the test compound at a specific dose can be achieved across the entire cell population or to an individual target cell. While often considered an advantage, the direct exposure of cells can also bring limitations. To be relevant and translatable to in vivo, this delivered concentration should be consistent with the estimated in vivo concentration in the target tissue (nervous system). In addition, it would be preferable if the compound were presented to the cells in a physiological formulation.

Syndromes

• Keep stuffed toys off the beds, and wash them weekly.
• Fainting or feeling light-headed
• Ingesting a drug or poison, especially by children
• Breathing in corticosteroids (from certain nose sprays)
• Head turns from side to side with sound at the level of the ear
• Breathing difficulty (from breathing in asphalt)
• What medications do you take?
• Fasting: greater than 95 mg/dL
• Urine tests to see if thiamine is passing through the urine
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It is also essential to calibrate system components on a regular basis during the course of the study allergy shots epinephrine buy discount periactin 4mg online, to verify that the performance of the several electronic and mechanical components has not changed over time allergy testing dogs blood discount periactin 4mg free shipping. Failure to calibrate the system on a regular basis means that there is no guarantee that the data collected at the beginning and end of the study are comparable allergy qld periactin 4mg discount. Failure to calibrate allergy testing johnson city tn purchase 4mg periactin with amex, combined with expressing the data in idiosyncratic (nonsense) units such as knob settings allergy medicine guide generic periactin 4mg with visa, precludes the opportunity to compare data across studies allergy symptoms with eyes discount periactin line, and deprives the informed reader of the ability to assess whether the data seem reasonable (an indication of whether the study was properly performed). Several investigators have explored the biological variables affecting vibration thresholds. There is a direct correlation between age and increased vibrotactile thresholds (Gerr et al. There is also a relationship between increased vibration threshold and greater height (Gerr et al. It appears that neither gender nor skin temperature (over the range usually encountered) has consistent effects on vibration thresholds. Differences in sensitivity between hands and feet have been reported, with lesser differences between sites of stimulation on the index finger or great toe. Increased vibration thresholds for the index finger were observed when tested using a pressure of 100 g per 1. In a study designed to assess the stability of vibration thresholds over time, 29 healthy females were tested using a two-interval forced-choice procedure and a routinely calibrated investigatordesigned system (Maurissen and Chrzan, 1989). Electrophysiological responses have been recorded after various forms of tactile stimulation. Stimuli have included taps, airpuffs, muscle flexion, vibration, and thermal stimulation (Hashimoto et al. Reproduced by permission of Elsevier from Maurissen and Chrzan (1989) Journal of Neurological Sciences 90, 325. When vibratory stimuli have been examined in the temporal domain, the recordings were separated into two components: a di- or triphasic response believed to be generated by multiple types of skin receptors and a phase-locked train of responses believed to be generated by Pacinian corpuscles (Hamano et al. Cortical somatosensory responses have also been recorded following vibratory stimulation of the fingers and palms (Snyder, 1992), as well as taps on fingernails (Pratt et al. In a series of experiments, Hashimoto and coworkers determined that air-puff stimuli can produce both peripheral nerve and brain somatosensory responses. The amplitudes and latencies of the peripheral (and integrated response) and central potentials were related to the intensity of the air-puff stimulation, and had a good correlation with the perceived intensities (Hashimoto et al. Additionally, somatosensory evoked magnetic fields have been recorded from the primary somatosensory cortex after air-puff stimuli, adding further evidence for the validity of the recordings (Forss et al. Thus, neurophysiological recordings can assess sensory responses to mechanical and electrical stimulation in both the peripheral and central portions of the nervous system in an objectively quantified manner. Both temperature and vibration are often assessed within a single study, a good strategy since damage to both large- (vibration) and small- (temperature) diameter fiber is thereby assessed. These systems usually take advantage of a Peltier element controlling the temperature of a metal plate in contact with the skin. When current is passed through the bimetallic junction of dissimilar elements, one side will cool and the other side will become heated, depending on the polarity of the current. Reversal of the temperature of the plate in contact with the skin is accomplished by reversal of the applied current. As with any sensory test, accurate calibration and reporting of the stimulus is required for comparison of the data between laboratories. As briefly mentioned above, neurophysiological evoked responses can also be determined after thermal stimulation (Pratt and Starr, 1986). The authors have shown that laser thermal stimulation of a human arm produces cortical responses that correspond to both the stimulus and subjective psychological response to the stimulus. Thermal stimulation of the hand, and subsequent cortical potentials, has been used to show that the adapting temperature (35 vs. Additionally, cooling of the hand has also been shown to evoke cortical potentials over the primary projection area for the hand on the postcentral gyrus (Duclaux et al. These methods have been used in conjunction with behavioral testing in humans and rats to assess thermal and pain sensitivity (Dotson, 1997; Katims, 1998; Kiso et al. Thus, while not as common as behavioral testing, several methods have been used to quantify physiological responses to thermal and pain stimulation. Several procedures were examined for stimulus presentation and data analysis, derived from standard psychophysical techniques and signal detection theory. The authors concluded that the forced-choice method was the best and the standard clinical procedure the worst of the tested procedures in detecting differences in sensitivity between body parts in normal subjects. Such methods represent an attempt on the part of clinical investigators to overcome the subjectivity inherent in the standard clinical neurological examination. A high correlation between such subjective symptoms and assessments of somatosensory function would not necessarily be expected, nor functions subserved by large versus small fibers be expected to be correlated, depending on the neurotoxic agent. The correlation between subjective clinical examination and more objective means of assessing sensitivity in various modalities is of particular interest. A study performed in patients receiving a standard hospital electrodiagnostic evaluation found a correlation between vibration thresholds, clinical neurological examination results, and nerve conduction velocities (Gerr et al. Neuropathy symptoms score, vibratory and cooling detection thresholds, nerve conduction velocity, and clinical neurological examination were compared in a study in diabetic patients (Dyck et al. The authors concluded that while most measures were associated as determined by regression analysis, the association was not high enough to be predictive. In a study in painters, there was no correlation between the results of qualitative assessment, neurological examination, and reported symptoms in individuals found to have higher vibration and temperature thresholds on objective assessment (Bove et al. The authors speculate that quantitative assessment may have detected early presymptomatic effects, or that lack of correlation may have resulted from individual differences in labeling intensity of reported events. These tests, typically performed with rodents, include such measures as forelimb and hind-limb grip strength, foot splay when dropped from a height, and ability to stay on a rotating rod (rotarod). Impairment of these measures may be due to impairment of somatosensory as well as motor function, including feedback from joints and muscles as well as skin. Such tests may be particularly sensitive to agents that produce both sensory and motor peripheral neuropathy, such as acrylamide (Gilbert and Maurissen, 1982; Moser et al. However, it is not possible to differentiate motor from sensory dysfunction using these procedures alone. If further delineation of sensory and motor function is desired, electrophysiological methodologies allow Table 3 Chemical Pesticides and related agents Effects Paresthesia, tingling, and impaired vibration, two-point discrimination, and depth discrimination Dysesthesia, paresthesia, and impaired touch, pain, temperature, vibration, and position sense References Anger et al. Tests for assessment of pain and/or temperature sensation have been used in the pharmaceutical industry since at least the 1950s. The jumping or flinching of a rodent when shock is applied to its feet is considered an indication of the intactness of cutaneous sensation. Pain is often evaluated by pinching the tail or foot to elicit a withdrawal response. Pain and/or temperature sensitivity are assessed by immersing the tail in hot water or placing the animal on a hot plate, and determining the latency of withdrawal. The frequency of head turns toward the flank in guinea pigs with flanks coated with synthetic pyrethroids has been used to quantify the tingling and burning sensations produced by these agents (McKillop et al. Operant methodology has been used extensively to assess sensory function in animals by psychophysical means (Cabe, 1982; Stebbins, 1970). The most commonly assessed somatosensory function in animals is undoubtedly vibration sensitivity. Vibration was delivered to a fingertip using a blunt needle attached to a computer-controlled system. The effects of acrylamide exposure have also been studied in monkeys using vibration sensitivity (Maurissen et al. Deficits were observed during dosing at both frequencies, and recovery was observed over the course of months after dosing ceased, outlasting effects such as weight loss and impairment on a visuomotor task. There was no deficit in the ability to detect an electrical stimulus, indicating sparing of small fibers. Nerve biopsy revealed mostly normalappearing nerve fibers, with a small proportion of fibers exhibiting changes typical of acrylamide toxicity. Vibration sensitivity was impaired for at least 6 months after cessation of dosing, at which point testing was discontinued. These changes were observed in the absence of changes in sensory or motor nerve conduction velocity. Somatosensory Neurotoxicity: Agents and Assessment Methodology Table 5 Agent Anticancer Cisplatin (cis-platinum) Therapeutic agents Effects Dysesthesia, paresthesia, and impaired vibration, position, and temperature sense References 329 Docetaxel Fludarabine and cytosine arabinoside Misonidazole Oxaliplatin Paclitaxel (taxol) Paresthesia Paresthesia Paresthesia and impaired vibration sense Cold hyperalgesia, peripheral neuropathy (sensory and motor) Dysesthesia, paresthesia, perioral numbness, and impaired vibration, position, pain, and temperature sense Paresthesia Paresthesia Pain, paresthesia, and impaired position sense Elderson et al. In Minamata, methylmercury was produced as a by-product in the manufacture of acetaldehyde and was discharged from an industrial plant into the Minamata River that fed into the Minamata Bay 330 Somatosensory Neurotoxicity: Agents and Assessment Methodology and surrounding areas of the Shiranui Sea. The methylmercury was taken up by marine biota and bioconcentrated in fish, which represented a substantial portion of the daily diet of people living in the region. Over 1000 people were eventually diagnosed as suffering from methylmercury poisoning. In two outbreaks of poisoning in Iraq, including a large episode in 1971, over 6000 people suffered severe neurological disease as the result of ingesting organomercury-treated seed grains (Bakir et al. In both Japan and Iraq, adult poisoning was characterized by motor and sensory impairment, including somatosensory dysfunction (Table 2). Somatosensory impairment is also a cardinal symptom of Minamata disease (Igata, 1993). The symptoms include decreased sensitivity in a glove-and-stocking pattern, although tingling and hyperesthesia have also been reported. Hypoesthesia or dysesthesia of mouth and limbs was still present as long as 30 years after poisoning, contributing to difficulty in carrying out daily activities such as eating, bathing, and dressing (Kinjo et al. These impairments grew relatively worse compared to unexposed individuals as a function of advancing age. Longer-term reassessments of somatosensory function in survivors of methylmercury poisoning in Minamata and other nearby fishing villages along the Shiranui Sea (Ninomiyaa et al. In the 1970s, hypoesthesias were found in > 70% of the residents of the town of Goshoura, which is located on the opposite shore of the Shiranui Sea from Minamata. However, histopathological samples of the sural nerve and electrophysiological studies did not detect peripheral nerve damage. The follow-up study of methylmercury poisoning cases in Minamata and Goshoura assessed somatosensory function quantitatively (Ninomiyaa et al. Touch-pressure sensation was evaluated using the ability to detect nylon filaments of different diameter, and thus applying different pressure to the skin. Two-point discrimination thresholds were also determined, that is, the smallest distance between two stimuli that was perceived as the touch of a single object. The group of individuals previously exposed to methylmercury had significantly higher touch-pressure thresholds than age-matched unexposed controls. The threshold distances for two-point discrimination of the mercury-exposed population were on average about two times higher than those of controls. If there was peripheral neuropathy in these patients when examined in the 1970s, it apparently resolved in the subsequent years, but the damage to somatosensory cortex persisted. The even distribution suggests that persistent impairments were a function of damage to the somatosensory cortex. Early symptoms included numbness and tingling in the extremities, with partial permanent paralysis at higher doses (Table 3). Subsequent outbreaks occurred in South Africa in 1937 and Morocco in 1959 as a result of contaminated cooking oil; over 10,000 individuals were affected in Morocco. Several extensive reviews of this topic are available (Abou-Donia, 2003; Craig and Barth, 1999; Glynn, 2007; Jokanovic et al. Methyl n-butyl ketone was increasingly used as a solvent until an outbreak of peripheral neuropathy in 1973 in a fabric plant in Ohio; it is now recognized to be more toxic than n-hexane. Both agents are metabolized by humans and animals to 2,5-hexanedione, the active neurotoxicant. Earliest symptoms of hexacarbon toxicity include sensory dysfunction in the hands and feet, including loss of touch, pinprick, vibration, and thermal sensation (Table 4). In the least-affected individuals, only sensory effects are present; at higher doses, toxicity progresses to distal weakness. In workers who were clinically normal on neurological examination, the amplitudes of sensory nerve action potentials from the sural and median nerves were decreased in workers exposed to n-hexane (Pastore et al. Partial or full recovery usually takes place after removal from the source of exposure, although signs and symptoms may at first intensify over the course of weeks or months. Hexacarbon toxicity may also result from inhalant abuse such as from glues containing n-hexane (Smith and Albers, 1997). Despite the well-known toxicity of n-hexane, cases of n-hexane-induced peripheral neuropathy continue to be reported around the world, such as in Turkey (Misirh et al. In California, between 1998 and 2000, cases of peripheral neuropathy were reported among workers in shops that repaired automobile brakes using a spray metal degreasing product containing n-hexane. Initial symptoms included numbness and tingling in the hands and feet that spread proximally to the forearms and waist. Neurological examination revealed diminished tendon reflexes and vibration and pinprick sensations in the lower third of the forearms and downward from the waist, and nerve conduction studies were consistent with peripheral neuropathy (Harrison et al. The concentration of neurofilament subunits in animals treated with n-hexane decreased in nerve tissues and increased in serum during the course of exposure to 2,5-hexanedione (Wang et al. Genetic polymorphisms may be important determinants of susceptibility to n-hexane.

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