As a developmental toxicologist, I am very interested in investigating the cellular and molecular mechanisms by which some forms of pollution alter normal development. I have two areas of research in my laboratory. The first research project is funded by the National Institutes of Health and examines the role beta-adrenergic receptor signaling plays in dioxin-induced dilated cardiomyopathy. The second research project investigates the incidence of imposex in dogwhelks (Nucella lapillus) along the Coast of Maine.
Project 1: Dioxin and Cardiac Beta-Adrenergic Receptor Signaling
TCDD, or dioxin, belongs to a family of structurally similar chemicals which include some polychlorinated biphenyls (PCBs). Significant amounts of these chemicals have entered the environment as contaminants and/or industrial products. Humans are involuntarily exposed to dioxin and PCBs through the diet and gradually accumulate them throughout their lifetime. The current average body burdens of dioxin and dioxin-like chemicals in humans in industrialized countries are very near levels known to cause developmental toxicity in laboratory animals. In the chick embryo, relatively low doses of dioxin injected into the egg soon after fertilization induce dilated cardiomyopathy and congestive heart failure (Walker and Catron, 2000). Mechanistic studies investigating the effects of dioxin exposure on the developing chick cardiovascular system demonstrated that the compact layer of the ventricular myocardial wall does not thicken in dioxin-exposed chick embryos (Walker et al., 1997; Walker and Catron, 2000), perhaps due to increased apoptosis and decreased myocyte proliferation that preceded a reduction in coronary artery number and size (Ivnitski et al., 2001).
My laboratory, in collaboration with Dr. M. K. Walker (a leading expert in the field of dioxin cardiovascular toxicology at the University of New Mexico), investigates the role that beta-adrenergic receptor (β-AR) signaling plays in dioxin-induced cardiovascular toxicity. β-ARs and their signal transduction systems give the normal heart the ability to increase its output by several-fold within a matter of seconds following activation by catecholamines. β-AR signaling also plays an important role in the processes of heart failure (for review see Port and Bristow, 2001), myocyte proliferation (Tseng et al., 2001; Sambrano et al., 2002), apoptosis (Communal et al., 1998; Zhu et al., 2003) and angiogenesis (Fredriksson et al., 2000). All of these processes have been shown to be altered by dioxin exposure. In addition, several studies have shown that acute exposure to overtly toxic doses of dioxin inhibits β-AR responsiveness (Brewster et al., 1987; Canga et al., 1988; Hermansky et al., 1988; Canga et al., 1993; Fan et al., 2000; Walker and Catron, 2000). Our work demonstrates that developmental exposure to relatively low doses of dioxin also reduces β-AR responsiveness, most likely by altering β-AR signal transduction upstream of adenylyl cyclase (Sommer et al., 2004).
It is possible that TCDD directly alters transcriptional expression of β-AR genes. TCDD is known to directly regulate the transcription of several genes by activation of two basic helix-loop-helix PAS transcription factors, the aryl hydrocarbon receptor (AhR) and the AhR nuclear translocator (ARNT) that, once activated, bind consensus DNA sequences (dioxin response elements, DREs) (for review see, Carlson and Perdew, 2002; Lai et al., 1996). We have identified five, three and two DREs in the 5’- flanking regions of the human, rat and mouse β1-AR genes, respectively. One DRE (~-385 bp, 5’ GGGCGTGCC 3’; core sequence underlined) is conserved in sequence and distance from the transcriptional start site in each of the human, rat and mouse β1-AR promoters, suggesting that this may represent a functional DRE (Sun et al., 2004). In addition, core DRE sequences are also present in the human and mouse β2-AR promoters (personal communication with T.R. Zacharewski). The sequence of the chicken β1-AR promoter is not known. On going studies in our laboratory are (1) cloning the 5’-flanking region of the chicken β1-AR promoter; (2) determining whether liganded AhR/ARNT directly binds putative DREs located in the promoter regions of β-AR genes of several species; and (3) determining whether AhR/ARNT/DRE binding is sufficient to transactivate β-AR gene expression.
Project 2: Imposex in dogwhelk snails (Nucella lapillus) along the Maine Coast
Tributyltin (TBT) is a biocide that in the past was used in antifouling paints to prevent buildup of barnacles and other encrusting organisms on the hulls of ships. Abundant field and laboratory data link TBT with the development of imposex (an irreversible masculinization of the female snail reproductive tract). Imposex includes development of male sex organs, notably a penis and vas deferens, which in some mollusc species prevent normal breeding activity and cause population decline. The occurrence of imposex is truly global, and has been reported from North America and Europe to Australia. However, limited data exists on the incidence of imposex in marine snail populations of the Maine coast. The only study that investigated this was conducted by Miller and Pondick (1984) which reported the presence of imposex in 94% of female dogwhelk snails (Nucella lapillus) sampled from New Harbor, Maine, and no imposex at Eastport or Pemaquid Point, Maine. A significant amount of data does exist for tissue concentrations of TBT and the resulting effects in blue mussels (Mytilus edulis) in Maine (Pageet al., 1995; 1996). This work demonstrated that although Maine is a state with a low human population density and relatively unpolluted coastline, moderate to high levels of TBT exist at many sites. The first objective of my work was to determine whether current levels of imposex in dogwhelk snail populations have change from those reported by Miller and Pondick in 1984, and to assess additional sites not yet surveyed. Results generated by Bates students working with me indicate that imposex of moderate frequency and intensity is found in multiple sites on the Maine Coast and that the incidence of imposex in Maine remained steady from the mid-1980’s to the late 1990’s and is now improving.
Current objectives of this research include verifying that TBT concentrations in the snails do in fact correlate with the presence of imposex. This work is being done in collaboration with Dr. Rachel Austin at Bates College, and has received NSF funding to purchase a GC-MS and GC-FID. Future work will investigate the mechanism(s) by which TBT induces imposex. Several possibilities exist and it is known that TBT increases testosterone levels in female snails. However, no definitive study has been done to explain how TBT increases testosterone concentrations and subsequently imposex.
Brewster, D. W., Matsumura, F., and Akera, T. (1987). Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on guinea pig heart muscle.Toxicol. Appl. Pharmacol. 89(3), 408-417.
Canga, L., Levi, R., and Rifkind, A. B. (1988). Heart as a target organ in 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity: Decreased β-adrenergic responsiveness and evidence of increased intracellular calcium. Proc. Natl. Acad. Sci. 85, 905-909.
Canga, L., Paroli, L., Blanck, T. J. J., Silver, R. B., and Rifkind, A. B. (1993). 2,3,7,8-Tetrachlorodibenzo-p-dioxin increases cardiac myocyte intracellular calcium and progressively impairs ventricular contractile responses to isoproterenol and to calcium in chick embryo hearts. Mol. Pharmacol. 44, 1142-1151.
Carlson, D. B., and Perdew, G. H. (2002). A dynamic role for the Ah receptor in cell signaling? Insights from a diverse group of Ah receptor interacting proteins. J. Biochem. Mol. Toxicol. 16(6), 317-325.
Communal, C., Singh, K., Pimentel, D. R., and Colucci, W. S. (1998). Norepinephrine stimulates apoptosis in adult rat ventricular myocytes by activation of the beta-adrenergic pathway. Circulation 98(13), 1329-1334.
Fan, L., Ovadia, M., Friedman, D. M., and Rifkind, A. B. (2000). Ventricular preexcitation sensitive to flecainide in late stage chick embry ECGs: 2,3,7,8-tetrachlorodibenzo-p-dixoin impairs inotropic but not chronotropic or dromotropic responses to isoproterenol and confers resistance to flecainide. Toxicol. Appl. Pharacol. 166, 43-50
Fredriksson, J. M., Lindquist, J. M., Bronnikov, G. E., and Nedergaard, J. (2000). Norepinephrine induces vascular endothelial growth factor gene expression in brown adipocytes through a β-adrenoreceptor/cAMP/protein kinase A pathway involving src but independently of erk1/2. J. Biol. Chem. 275(18), 13802-13811.
Hermansky, S. J., Holcslaw, T. L., Murray, W. J., Markin, R. S., and Stohs, S. J. (1988). Biochemical and functional effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the heart of female rats.Toxicol. Appl. Pharmacol. 95(2), 175-184
Ivnitski, I., Elmaoued, R. and Walker, M. K. (2001). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibition of coronary development is preceded by a decrease in myocyte proliferation and an increase in cardiac apoptosis. Teratology 64, 201-212.
Lai, Z. W., Pineau, T., and Esser, C. (1996). Identification of dioxin-responsive elements (DREs) in the 5′ regions of putative dioxin-inducible genes. Chem. Biol. Interact. 100(2), 97-112.
Miller, E. R. and Pondick, J. S. (1984). Heavy metal levels in Nucella lapillus (Gastropoda: Prosobranchia) from sites with normal and penis-bearing females from New England. Bull. Environ. Contam. Toxicol.33, 612-620
Page, D. S., Dassanayake, T. M., and Gilfillan, E. S. (1995). Tissue distribution and depuration of tributyltin for field-exposed Mytilus edulis. Marine Environ. Res. 40(4), 409-421.
Page, D. S., Dassanayake, T. M., and Gilfillan (1996). Relationship between tissue concentrations of tributyltin and shell morphology in field populations of Mytilus edulis. Bull. Environ. Contam. Toxicol. 56, 500-504.
Port, D. J., and Bristow, M. R. (2001). Altered -adrenergic receptor gene regulation and signaling in chronic heart failure. J. Mol. Cell. Cardiol. 33, 887-905.
Sambrano, G. R., Fraser, I., Han, H., Ni, Y., O’Connell, T., Yan, Z., and Stull, J. T. (2002). Navigating the signaling network in mouse cardiac myocytes. Nature 420, 712-714.
Sommer, R. J., Hume, A. J., Ciak, J. M., VanNostrand, J. J., Friggens, M. and Walker, M. K. (2004). 2,3,7,8-Tetrachlorodibenzo-p-dioxin Decreases Chick Embryo Heart Chronotropic Response to Isoproterenol but not to Agents Effecting Downstream Signals of the Beta-Adrenergic Receptor. Submitted to Toxicol. Sci.
Sun, Y., Boverhof, D. R., Fielden, M. R., and Zacharewski T. R. (2004). Comparative analysis of dioxin regulatory elements in human, mouse and rat genomic sequences. Toxicologist, 78(1-S), 604 (Abstract).
Tseng, Y. T., Kopel, R., Stabila, J. P., McGonnigal, B. G., Nguyen, T. T., Gruppuso, P. A., and Padbury, J. F. (2001). Beta-adrenergic receptors (betaAR) regulate cardiomyocyte proliferation during early postnatal life. FASEB J. 15(11), 1921-1926.
Walker, M. K., and Catron, T. F. (2000). Characterization of cardiotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related chemicals during early chick embryo development. Toxicol. Appl. Pharmacol. 167, 210-221.
Walker, M. K., Pollenz, R. S., and Smith, S. M. (1997). Expression of the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator during chick cardiogenesis is consistent with 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced heart defects. Toxicol. Appl. Pharmacol. 143, 407-419.
Zhu, W. Z., Wang. S. Q., Chakir, K., Yang, D., Zhang, T., Brown, J. H., Devic, E., Kobilka, B. K., Cheng, H., and Xiao, R. P. (2003). Linkage of beta1-adrenergic stimulation to apoptotic heart cell death through protein kinase A-independent activation of Ca2+/calmodulin kinase II. J. Clin. Invest. 111(5), 617-625.
Research Honors and Awards
1996-1998 Samuel C. Johnson Distinguished Fellowship for Graduate Research
1995 Society of Toxicology Annual Meeting Graduate Student Travel Award