In my lab we have have used the sea urchin embryo model to examine the role of serotonin in early embryogenesis prior to development of a nervous system. We have also examined the potential role of catecholamines in heart development in the chicken embryo model. Finally, 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 established a research program in developmental and reproductive toxicology to examine mechanisms of embryo toxicity of various neurochemicals, endocrine disruptors (including bisphenol A), heavy metals, and silver nanoparticles.
1) Role of Serotonin and Catecholamines in Early Embryogenesis
Several chemicals (neurotransmitters) that 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.
a) Serotonin initiates gastrulation cell movements in sea urchin embryos
We have detected serotonin, a local hormone and neurotransmitter, in sea urchin embryos that have not yet developed organs. Serotonin levels peak immediately prior to gastrulation, a cell movement process that places them in locations where they will eventually develop into different tissue types, and serotonin is localized to cells that will initiate these movements. Furthermore, we have identified a gene that codes for a serotonin synthetic enzyme (PAH/TPH), and inhibition of this enzyme blocks serotonin synthesis and gastrulation. In vertebrates, serotonin functions by first binding to one of 7 receptor types. We have shown that six of these, Types 2-7, are present in the sea urchin genome. Three of these receptor Types (2, 5, and 7) are expressed in gastrula embryos, and inhibitors of Types 2 and 7 receptors block the initiation of gastrulation. Finally, we have been able to rescue initial gastrulation events in embryos cultured in the presence of a serotonin synthesis inhibitor by co-addition of serotonin or molecules normally activated by serotonin receptor Types 2 or 7. From these studies we have hypothesized that serotonin initiates gastrulation cell movements by first binding to Type 7 and/or 2 serotonin receptors.
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 have examined the potential role of dopamine and the other catecholamines, epinephrine and norepinephrine, in heart development. For these studies we utilized the chicken embryo model, since heart development in birds and humans is comparable, and have developed a unique in vitro embryo culture technique. We showed 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 that do not form a heart when cultured in the absence of dopamine. Furthermore, dopamine synthesis inhibitors block heart tube formation in whole in vitro cultured embryos. Since the other catecholamines, norepinephrine and epinephrine, can be synthesized from dopamine, we proceeded to determine if either of these was the actual catecholamine involved in heart formation. From these studies we have obtained preliminary evidence that norepinephrine signaling through α-adrenergic receptors stimulates expression of the transcription factor, Gata4, triggering heart tube formation.
2) Developmental and Reproductive Toxicology Studies
Most recent studies: effects of silver nanoparticles on mouse embryogenesis
In this collaborative project with the Center for Devices and Radiological Health, U.S. Food and Drug Administration, we examined the potential effects of silver nanoparticles on mouse embryos following exposure of pregnant females. These small particles are present in 40% of all nanotechnology-based consumer products from socks to cell phones to baby pacifiers and blood catheters. They are so small that they can easily penetrate cells, and little is known about their toxicity to the developing embryo, or even whether they can pass through the placenta and into the embryo. In our studies we exposed pregnant mice to 10 nm and 50 nm silver nanoparticles during the time in which their embryos are forming different organs, and examined silver levels, cellular and subcellular location of silver nanoparticles, and anatomy of adult and embryonic tissues. We showed that high levels of silver nanoparticles accumulate in the adult liver and spleen and in placental tissues but do not reach the developing embryo. Our electron microscopic and hyperspectral light microscopic studies have shown that placental cells accumulate particles that do not get transferred to blood vessels that connect with the embryonic circulation. However, we also showed that embryos of treated mothers exhibited stunted growth. We have postulated that this is due to silver nanoparticle-induced interference of placental nutrient transport to fetal blood vessels.
Austin C.A., Hinkley G.K., Mishra A.R., Zhang Q., Umbreit T.H., Betz M.W., E Wildt B., Casey B.J., Francke-Carroll S., Hussain S.M., Roberts S.M., Brown K.M. and Goering P.L. (2016) Distribution and accumulation of 10 nm silver nanoparticles in maternal tissues and visceral yolk sack of pregnant mice, and a potential effect on embryo growth. Nanotoxicology 10, 654-661.
Austin C.A., Umbreit T.H., Barber D.S., Dair B.J., Francke-Carroll S., Fenwick A., Saint-Louis M.A., Hikawa H., Sievein K.N., Goering P.L. and Brown K.M. (2012) Distribution of intravenously-injected silver nanoparticles in pregnant mice and developing embryos. Nanotoxicology 6, 912-922.
Carroll K.N., Scully T.A., Anitole-Misleh K.G., Johnson D.E. Brown K.M. (2010) Serotonin initiates gastrulation in the sea urchin. Dev. Biol. (suppl), 118.
Scully T., Carroll K.N., Brown K.M. (2009) A serotonin-mediated signaling mechanism initiates cell movements during sea urchin gastrulation. Integr. Comp. Biol. 49, e191-e332.
Anitole, KG. and Brown, K.M. (2004) Developmental regulation of catecholamine levels during sea urchin embryo morphogenesis. 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 ovariectomized 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.M. 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.