Ken M. Brown Professor of Biology and of Genetics Developmental Biology Department of Biological Sciences The George Washington University Lisner Hall 332, 2023 G Street, NW Washington, D.C. 20052 Office: (202) 994-6193 Fax: (202) 994-6100 E-Mail: kmb@gwu.edu Dept E-mail : biology@gwu.edu

1) Role of Serotonin and Catecholamines in Early Embryogenesis

Several chemicals (neurotransmitters) which mediate nerve cell communication, including acetylcholine, serotonin, dopamine and norepinephrine, have been identified in both invertebrate and vertebrate embryos that have not yet developed a nervous system. The role of neurotransmitters in these preneural embryos is not understood. Results of studies from my lab and others suggest that these monoamines may regulate several basic developmental processes common to all animal embryos including cleavage, cell movements and cell shape changes, and cell differentiation (Brown and Anitole, 1993; Anitole and Brown, 2004).

In my lab we have used the sea urchin embryo model to examine the role of serotonin and catecholamines in early embryogenesis. We are also examining the potential role of catecholamines in heart development in the chicken embryo model.

a) Serotonin in early sea urchin embryogenesis

We have shown that chemicals which block central nervous system activity of serotonin in adult mammals will also inhibit sea urchin early gastrulation (Anitole et al., 1988b), a process which involves the inward folding of a layer of cells which will eventually form the walls of the gut tube. Furthermore, this inhibition can be reversed with serotonin and other agents which affect cyclic AMP and calcium ion levels (Anitole et al., 1988a). Binding of radiolabelled serotonin to whole cells (Brown and Shaver, 1989) and subcellular fractions (Brown and Shaver, 1987) has demonstrated the presence of intracellular serotonin binding sites in blastula and gastrula embryos and cell surface binding sites in post-gastrula embryos. From these studies and others we have postulated that serotonin intiates cell movements during early sea urchin gastrulation by interacting with intracellular receptors which are coupled to the cell cytoskeleton, either directly or via a signal transduction system involving cAMP and calcium ions.

We have completed an extensive analysis of the content of serotonin and catecholamines, and their precursors and metabolites in blastula, gastrula and post-gastrula embryos using high performance liquid chromatography with electrochemical detection (HPLC-EC) (Brown and Anitole, 1998; Anitole and Brown, 2004). These studies are the first to definitively identify monoamines in these early embryos and support a role for serotonin (and norepinephrine) in the gastrulation process as suggested from our previous inhibitor and binding studies. We have also shown by RT-PCR analyses that the messenger RNA (mRNA) for tryprophan hydroxylase (serotonin synthetic enzyme) increases in embryos immediately prior to an increase in serotonin at gastrulation. We are currently examining the intraembryonic location of the mRNAs for tryptophan hydroxylase and the serotonin receptor, a molecule that mediates the action of serotonin in neurons, by in situ hybridization. These studies should allow us to determine if, as in mammalian neurons, the cells that synthesize serotonin are different from those that respond to it.

b) Role of catecholamines in heart cell differentiation and heart morphogenesis

Little is known about the molecules that regulate the formation of a heart, the first organ to develop in vertebrate embryos. We are currently examining the potential role of dopamine and the other catecholamines, epinephrine and norepinephrine, in heart development. For these studies we are utilizing the chicken embryo model, since heart development in birds and humans is comparable, and have developed a unique in vitro embryo culture technique. We have shown that dopamine is present in early (gastrula stage) embryos and that dopamine induces differentiation of heart cell tissue in vitro in pieces of gastrula embryos which do not form a heart when cultured in the absence of dopamine. Furthermore, catecholamine synthesis inhibitors block heart development in whole in vitro cultured embryos (Kirk et al. 1998; Kirk et al., Role of catecholamines in heart morphogenesis, in prep.). From these studies we have postulated that dopamine regulates heart development. Since the other catecholamines, norepinephrine and epinephrine, can be synthesized from dopamine, we are currently investigating whether either of these substances is necessary for heart development. We are also examining potential links between various heart-specific transcription factors (proteins that modulate gene expression) and the activation of a catecholaminergic pathway during early chicken embryogenesis.

2) Developmental and Reproductive Toxicology Studies

In collaboration with other researchers in the G.W.U. Forensic Sciences Department, Catholic University Physics Department and the U.S. Food and Drug Administration, I have established a research program in developmental and reproductive toxicology. We have examined the mechanisms of heavy metal-mediated embryotoxicity (Papaconstantinou et al., 2003b) and of cocaine- (Burin et al., 1991; Brown and Burin, 1993) and electromagnetic radiation- (Litovitz et al., 1994; 1997) induced neural tube defects in in ovo and in vitro cultured chicken embryos. We have also examined the role of stress proteins and intermediate filaments in stress-mediated skeletal defects in rat embryos in vivo an in in vitro cultures (Fisher et al., 1995; 1996).

We are currently examining the potential estrogenic or antiestrogenic activity of a number of environmental contaminants and plant derived chemicals (Papaconstantinou et al. 2002a; 2002b). These chemicals can potentially disrupt development, growth, sexual differentiation and reproductive function. We have developed an in vivo mouse assay for these substances that is based on their ability to alter uterine morphology and affect levels of heat shock (stress) proteins in the uterus and dopamine levels in the brain.

We are particularly interested in the environmental estrogen, bisphenol A. This chemical is used in the manufacture of several types of plastics. Concern about potential exposure to humans is based on studies indicating leaching from food can linings, baby bottles, dental sealants and medical devices. Using our mouse uterine model, we were the first to show that the effects of bisphenol A on uterine morphology and gene expression are mediated by the estrogen receptor (Papaconstantinou et al., 2000; 2001) and also by non-receptor pathways that involve the activation of protein kinase C (Papaconstantinou et al., 2003a). We are currently studying the effects of bisphenol A on morphology and gene expression profiles in cultures of 16-day rat embryo hippocampal brain cells. At this stage of development the brain is sex neutral. By 17 days, rat embryo brains begin to be exposed to sex steroids (estrogen and progesterone) which result in male- and female-specific neuronal pathways. Although bisphenol A is a weak estrogen in the uterine model, our preliminary results suggest that it is a more potent estrogen in the embryonic brain.

Future Studies

In my laboratory, we have recently begun to examine the effects of nanoparticles on embryonic development and on in vitro cultures of adult hippocampal brain cells. Nanoparticles are chemical structures, often crystalline in form, that may be as small as an individual protein molecule, and are currently being mass-produced for use in extremely miniaturized electrical circuits, in medicine, and as tools for cell and molecular biologists. These particles can penetrate cells and their potential toxic effects on biological systems are unknown.

We will also continue to decipher the cellular and molecular mechanisms of serotonin-, catecholamine- and estrogen-mediated cell movement, heart formation and brain cell differentiation, respectively, in developing embryos. While these studies should tell us more about how these common developmental processes are regulated in normally developing embryos, they have broader implications regarding the potential for pharmaceuticals and environmental estrogens to disrupt these processes.

Anitole, KG. and Brown, K.M. (2004) Developmental regulation of catecholamine levels during sea urchin embryo morhogenesis. Comp. Biochem. Physiol. Part A (137, 39-50).

Anitole K.G., Butler C.L., Lappas N.T. and Brown K.M. (1988b) Chlorpromazine-sensitive developmental processes in the sea urchin, Lytechinus pictus. 2. Effects of neuroactive agents on the susceptibility of the gastrulation process to chlorpromazine. Comp. Biochem. Physiol. 90C, 55-60.

Anitole K.G., Stahle P.L., Ridenour C.S., Lappas N.T. and Brown K.M. (1988a) Chlorpromazine-sensitive developmental processes in the sea urchin, Lytechinus pictus. 1. Inhibition of cleavage, gastrulation, and primary mesenchyme cell differentiation. Comp. Biochem. Physiol. 90C, 47-53.

Brown K.M. and Anitole K.G. (1993) Serotonin in early sea urchin embryogenesis. Trends in Comparat. Biochem. Physiol. 1, 281-288.

Brown K.M. and Anitole K.G. (1998) Serotonin and early sea urchin embryogenesis: induction of serotonergic neurons. Dev. Biol. 198, 190.

Brown K.M. and Burin G.J. (1993) Cocaine-mediated disruption of microfilament integrity within neural fold neuroepithelial cells of chick embryos cultured in vitro. Toxic. in Vitro 7, 285-289.

Brown K.M. and Shaver J.R. (1987) Subcellular distribution of serotonin binding sites in blastula, gastrula, prism, and pluteus sea urchin embryos. Comp. Biochem. Physiol. 87C, 139-148.

Brown K.M. and Shaver J.R. (1989) Serotonin binding to blastula, gastrula, prism, and pluteus sea urchin embryo cells. Comp. Biochem. Physiol. 93C, 281-285.

Burin G.J., Al-Ghaith L.K., Anitole K.G., Barber M.K. and Brown K.M. (1991) Investigation of the developmental toxicity of cocaine in in vitro cultured chick embryos: correlation of effects with intraembryonic drugs levels. Toxic. in Vitro 5, 285-293.

Farrell J.M., Litovitz T.L., Penafiel M., Montrose C.J., Doinov P., Barber M., Brown K.M. and Litovitz T.A. (1997) The effect of pulsed and sinusoidal magnetic fields on the morphology of developing chick embryos. Bioelectromagnetics 18, 431-438.

Fisher B.R., Heredia D.L. and Brown K.M. (1995) Induction of hsp 72 in heat-treated rat embryos: a tissue specific response. Teratology 52, 90-100.

Fisher B.R., Heredia D.L. and Brown K.M. (1996) In vitro heat shock produces alterations in cytoskeletal proteins in cultured rat embryos. Teratogen. Carcinogen. Mutagen. 16, 49-64.

Litovitz T.A., Montrose C.J., Doinov P., Barber M. and Brown K.M. (1994) Superimposing spatially coherent electromagnetic noise inhibits field-induced abnormalities in developing chick embryos. Bioelectromagnetics 15, 105-113.

Kirk D.K., Kennison S, and Brown K.M. (1998) Dopamine and chicken embryo heart development. Dev. Biol. 198, 210.

Papaconstantinou, A.D., Brown, K.M., Fisher, B.R. and Goering, P.L. (2003b) Stress
protein synthesis induced by mercury, cadmium and arsenic in chick embryos. Birth
Defects Res. C (Part B) 68, 456-464.

Papaconstantinou A.D., Brown K.M., Lappas N.T., Fisher B.R. and Umbreit T.H. (1998) Estrogenicity and heat shock proteins: bisphenol A. Tox. Sci. 42, 175.

Papaconstantinou A.D., Fisher B.R., Umbreit T.H. and Brown K.M. (2002a) Increases in mouse uterine heat shock protein levels are a sensitive and specific response to uterotrophic agents. Environ. Health Perspect. 110, 1207-1212.

Papaconstantinou A.D., Goering P.L., Umbreit T.H. and Brown K.M. (2003a) Regulation of uterine hsp90α, hsp72 and HSF-1 expression in B6C3F1 mice by β-estradiol and bisphenol A: Involvement of the estrogen receptor and protein kinase C. Toxicol. Lett. 144, 257-270.

Papaconstantinou A., Umbreit T.H., Fisher B.R., Goering P.L., Lappas N.T. and Brown K.M. (2000) Bisphenol A - induced increase in uterine weight and alterations in uterine tissue morphology in the ovarectomized B6C3F1 mouse: role of the estrogen receptor. Tox. Sci. (56, 332-339).

Papaconstantinou A.D., Umbreit T.H., Goering P.L. and Brown K.M. (2002b) Effects of 17α-methyltestosterone on uterine morphology and heat shock protein expression are mediated through estrogen and androgen receptors. J. Steroid Biochem. Molec. Biol. 82, 305-314.

Shah M., Brown K. and Smith C. (2003) The gene encoding the complement protein, SpC3, is expressed in embryos and can be induced by bacteria. Dev. Comp. Immunol. 27, 529-538.

Silbergeld E.K., Flaws J.A. and Brown K.M. (2001) Organizational and activational effects of estrogenic endocrine disrupting chemicals. CSP Reports in Public Health 18, 489-494.

Undergraduate:
BiSc 014 - Introductory Biology: Cells and Molecules (Spring semester)
BiSc 114 - Developmental Biology (Fall semester)
BiSc 115 - Experimental Developmental Biology (Spring semester, even years)
BiSc 171; BiSc 173 - Undergraduate Research; Independent Study (Fall and Spring semesters)

Carlye Austin (Ph.D. candidate, neuro- and developmental toxicology of titanium- and carbon-based nanoparticles, caustin@gwu.edu)

Sofia Pagedas (M.S. candidate, Bisphenol A-mediated alteration in gene activity in primary cultures of rat hippocampal brain cells, spagedas@gwu.edu)