Spring 2005 Symposium Graduate Student/Postdoc Poster Awards

May 24, 2005

 

 

 

1st Place   ($ 300)       

Kim Barnett, University of Maryland Program in Toxicology

 

2nd Place   ($ 250)      

Rosemary Schuh, PhD, University of Maryland Program in Toxicology

 

3rd Place  ($ 200)

            Christopher Toscano, MS, PhD, Johns Hopkins University

 

 

 

Spring 2005 Symposium Graduate Student/Postdoc Poster Abstracts

 

 

Kim Barnett

 

The Aryl Hydrocarbon Receptor (AhR) Regulates Ovarian Follicle Growth In Vitro

Barnett KR; Tomic D; Flaws JA.  University of Maryland School of Medicine, Baltimore, MD

 

The AhR plays an important role in mediating the toxicity of various environmental toxicants that cause adverse effects on the development and function of the female reproductive tract. Studies using AhR-deficient (AhRKO) mice have shown that the AhR has an important physiological role in the mouse ovary. Previous studies in our lab have demonstrated that AhRKO ovaries have a decreased number of antral follicles compared to wild-type (WT) ovaries. Since our previous studies also indicate that AhR deficiency does not affect atresia (follicle death via apoptosis) of antral follicles, the purpose of these studies was to determine whether AhR deficiency reduces follicle numbers by slowing follicular growth. Further, since antral follicles produce estradiol (E2) and E2 is required for normal follicular growth, these studies also tested whether AhR deficiency results in decreased synthesis of E2 by antral follicles. To test these hypotheses, antral follicles were isolated from AhRKO and WT ovaries and cultured for 168 hours. During culture, follicle growth was assessed by daily measurements of follicular diameter. After culture, media was collected and E2 levels were measured using an enzyme-linked immunoassay (ELISA). AhRKO and WT ovaries were also subjected to measurements of proliferation using immunohistochemistry (IHC) for proliferating cell nuclear antigen (PCNA) antibody. Our results show that WT follicles grew significantly larger than AhRKO follicles by 168 hours of culture (WT: 615.5±17.15mm; AhRKO: 489±17.03mm; p<0.001; n=3 mice per genotype, 10 follicles per mouse). The results also show that WT follicles produced significantly more E2 compared to AhRKO follicles (WT: 2463±508 pg/ml, n=15 follicles; AhRKO: 971±316 pg/ml, n=9 follicles; p=0.007). Further results from IHC show that AhRKO follicles had less PCNA staining in granulosa cells compared to WT follicles. These data suggest that in addition to mediating toxicity of environmental chemicals, the AhR is an important regulator of ovarian follicle growth and E2 production. [Supported by NIH grants GM072195-01, HD38955, and R25-GM55036]

 

 

Rupesh Gupta

 

Methoxychlor Inhibits Expression of Antioxidant Enzymes in the Mouse Ovary

Gupta RK, Miller KP, Tomic D and Flaws JA. Program in Toxicology, University of Maryland-School of Medicine, Baltimore, MD, USA

 

Females are born with a finite number of primordial follicles, of which a small fraction reaches the antral stage. Antral follicles are responsible for releasing an egg for fertilization and maintaining cyclicity. In vivo studies with the organochlorine pesticide methoxychlor (MXC) have shown that antral follicles are the primary targets of MXC exposure. Specifically, MXC exposure decreases the number of antral follicles and increases the percentage of antral follicles undergoing atresia (cell death via apoptosis). While different pathways lead to toxicant-induced cell death, oxidative stress is known to cause apoptosis in non-reproductive and reproductive tissues. Certain toxicants produce reactive oxygen species, which are detoxified by antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT). Thus, this work tested the hypothesis that MXC inhibits the expression of selected antioxidant enzymes in antral follicles. To test this hypothesis, 39-day old CD-1 mice were dosed with either sesame oil (control) or MXC (32 or 64 mg/kg/day) for 20 days. After treatment, ovaries were collected and antral follicles were isolated from the ovaries and subjected to real time polymerase chain reaction for measurement of mRNA levels of SOD, GPX, and CAT. The results indicate that MXC significantly decreases mRNA expression as compared to controls of SOD (control=2.98 ± 0.30 genomic equivalents (ge); MXC 32 mg/kg/day=0.94 ± 0.08 ge; MXC 64 mg/kg/day=1.28 ± 0.16 ge; n=3; p £ 0.003), GPX (control=2.36 ± 0.48 ge; MXC 32 mg/kg/day=0.90 ± 0.03 ge; MXC 64 mg/kg/day=1.09 ± 0.10 ge; n=3; p £ 0.05), and CAT (control=2.02 ± 0.24 ge; MXC 32 mg/kg/day=0.98 ± 0.05 ge; MXC 64 mg/kg/day=1.13 ± 0.07 ge; n=3; p £ 0.01). Collectively, these data indicate that MXC inhibits the expression of SOD, GPX, and CAT in antral follicles. Therefore, it is possible that MXC may cause atresia of ovarian antral follicles by inducing oxidative stress through inhibition of SOD, GPX, and CAT detoxifying pathways. [Supported by NIH HD38955, T32 ES07263, and a Colgate Palmolive Fellowship]

 

 

Rosemary Schuh

 

The Organochlorine Pesticide Methoxychlor Alters Brain Mitochondrial Respiration, H2O2 Production and Calcium/cAMP Response Element Binding Protein Levels.

R.Schuh1,2 T. Kristian1, J. Flaws2 and G. Fiskum1,2  Department of  1Anesthesiology, 2Epidemiology and Experimental Therapeutics, Program in Toxicology, University of Maryland School of Medicine, Baltimore, MD, USA

 

Methoxychlor, an organochlorine insecticide with endocrine disruptive properties has been demonstrated to affect the reproductive system. However, this environmental contaminant is also implicated in decreasing antioxidant enzyme activity and increasing hydrogen peroxide production resulting in oxidative stress. Phosphorylation of the Ca2+/cAMP response element binding protein (CREB) has been demonstrated in many studies to increase in response to oxidative stress. In the current study, we tested the hypothesis that methoxychlor inhibits mitochondrial respiration, increases mitochondrial hydrogen peroxide production and alters the phosphorylation state of mitochondrial CREB. Mitochondria isolated from rat brains were exposed in vitro to methoxychlor (0-10 mg/ml). In addition, mitochondria were isolated from brains of mice chronically exposed in vivo to methoxychlor (0-64 mg/kg/day) for 20 days by intraperitoneal injection. In vitro methoxychlor exposure inhibited state 3 (ADP-stimulated) O2 consumption and respiration-dependent H2O2 production was stimulated, both in a dose-dependent manner. ADP-stimulated O2 consumption was inhibited in isolated mitochondria from mice exposed in vivo to methoxychlor but without stimulation of H2O2 production suggesting a compensatory mechanism had been invoked. Analysis by ELISA demonstrated a dose-dependent increase in phosphorylated CREB in the mitochondrial lysates exposed in vitro to methoxychlor in the absence or presence of respiratory substrates. These results suggest that methoxychlor exposure causes mitochondrial metabolic stress (in vitro and in vivo) and oxidative stress (in vitro only). In vitro methoxychlor also increases mitochondrial pCREB but in a manner that does not require mitochondrial H2O2 generation. [Supported by NIH grants ES07263 (R.S.) and NS34152 (G.F.), and USAMRMC grant DAMD 17-99-1-9483 (G.F.)]

 

 

Christopher Toscano

 

Temporal Parameters of Environmental Enrichment-Induced Cognitive Enhancement in a Rodent Model of Lead Neurotoxicity. CD Toscano, JL McGlothan, JR Moss, TR Guilarte, Johns Hopkins University, Baltimore, MD.

 

Environmental enrichment (EE), a non-pharmacological therapy that combines social interaction with a complex living environment, reverses molecular and cognitive deficits observed in a rodent model of lead intoxication (Guilarte et al, Ann. Neurol., 53:50, 2003). In order to translate this finding to the human condition, it is important to further understand the temporal parameters of this therapy. We tested whether the benefit of EE on cognitive function persists in adult rats after EE was removed and if a critical window exists for the application of this therapy.  Rats were exposed to 0 or 1500 ppm lead acetate from conception until postnatal day (PN) 21 and then housed singly in standard rat cages (isolated) or in groups of 8 (enriched) in multi-level cages that contained toys until PN79. To test if the benefits of EE are long lasting, rats were raised in enrichment cages from PN21 until PN50 and then transferred to isolated cages until PN79 (permanence). To determine if a critical window existed for the benefit of the intervention, animals were placed in EE from PN50 to PN79. In all studies, spatial learning was assessed at PN79. Blood and hippocampal lead levels were elevated in lead exposed rats, however, no significant Pb2+-exposure effect was observed on the acquisition of the task. A significant housing effect was observed on the acquisition, probe and cue tests with rats currently receiving EE (enriched and critical window) performing significantly better on all three tasks. Nearly twice as many rats in the isolated and permanence groups exhibited a place strategy in the cue test which contributed to the significantly elevated latency in the cue test. In summary, these studies could not detect a significant cognitive deficit in lead exposed rats at PN79, which could be due to the degree of difficulty of the task. Further, EE is effective in enhancing cognitive performance but this benefit is lost after cessation of EE. [Supported by NIEHS grant # ES006189 to TRG]

 

 

Meg Whittaker

 

Induction of Oxidative Stress in Response to Ingestion of Lead, Cadmium and Arsenic Mixtures

M. Whittaker, M. Lipsky, G. Wang, X. Chen, and B.Fowler.  Toxicology Program, University of Maryland, Baltimore.

 

Human populations are commonly exposed to mixtures of chemicals.  To date, relatively few studies have examined potential interactive effects using a statistical factorial design.  Multiple drinking water studies were performed to test the hypothesis that exposure to arsenic, lead, or cadmium (or their combinations) for 30, 90, or 180 days at lowest-observed-effect levels (LOELs) results in increased levels of oxidative stress in the kidney, which is a target organ for trace element-induced toxicity.  Male Sprague-Dawley rats were exposed to lead, cadmium, arsenic, or mixtures of these three trace elements for 30, 90, or 180 days via drinking water.  Oxidative stress levels (as measured by increases in kidney carbonyls) were generally increased at 30 days and decreased at 90 and 180 days.  At 30 and 180 days, cadmium appeared to attenuate carbonyl increases among mixture groups.  Among all treatment groups, increases in kidney carbonyls were lowest among the PbxCdxAs group at all three timepoints.  Cellular adaptation to trace element-induced oxidative stress is suggested by the attenuation of increases in kidney carbonyls at the 90 and 180 day timepoints.  Statistically significant increases in kidney glutathione levels (measured as nonprotein thiols) were measured after 30 and 180 days of exposure among most treatment groups, with some of the greatest increases measured among the four combination groups at the 30 day timepoint (96%-145% increase) and the 180 day timepoint (20%-70% increase).  In contrast, kidney non-protein thiols were statistically significantly decreased in 4 of 7 treatment groups after 90 days of exposure (28%-33% decrease).  These data demonstrate that low-level exposure to trace elements or their mixtures results in measurable increases in oxidative stress and upregulation of cellular defensive mechanisms. [Supported by U.S. EPA Star Grant R827161-01-0]

 

 

Mashael Al-Namaeh

 

Characterization of the nAChR in Rat Heart During Development and Regulation by Nicotine.  Al-Namaeh, M., , Das J.R., and Dávila-García, M.I. Department of Pharmacology, College of Medicine, Howard University, Washington. D.C.

 

Cigarette smoking during pregnancy increases the incidence of perinatal mortality and cardiovascular diseases. We know nicotine exposure alters neuronal nicotinic acetylcholine receptors (nAChRs) in cardiac vagal parasympathetic preganglionic neurons (cVPN) that project to cardiac parasympathetic ganglionic neurons (GPNs), which also contain nAChRs.  Therefore, our goal was to determine the identity of the nAChR subtypes in the heart, determine their developmental profile, and assess if they were regulated by prenatal nicotine.  Our working hypotheses were that 1) nAChRs will increase with developmental age and 2) that nicotine upregulates these receptors. We tested our hypotheses using [3H]EB receptor binding assays and [125I]EB binding and autoradiography. Pregnant rats received continuous infusions of saline or nicotine (4mg/Kg/day) from embryonic day 7 through birth. The results show that nAChRs are developmentally regulated with a peak at P7. The developmental profile, seems to be identical between control and nicotine exposed hearts tissues.  [3H]EB binding assays were performed in the presence of 15 nM A85380 or 200 nM cytisine. The data shows that in the whole heart, approximately 38% of nAChRs are β2-containing, since A85380 is selective for these receptors. The residual binding in the presence of A85380 (~62%) represent all non β2-containg receptors. Since cytisine displaces all the nAChRs except α3β2 or α3β4 or α3β4α5,  its displaced binding (~43%) also represents the β2-containing receptors, while the residual binding (~57%) represent β4-containing receptors, Since there was no difference in the levels of residual binding between A85380 and cytisine, therefore, it is unlikely any α3β2 receptors are present in the rat heart. The data demonstrates that there are at least two potential types of nAChRs in rat heart, in addition to the known α7 receptors (Ji et al., 2002), a β2-containing receptor, but not an α3β2, and high levels of a low affinity α3β4* receptor. Furthermore, these receptors are upregulated by nicotine only at E18. Thus, during the critical period of rapid development and synaptogenesis, prenatal nicotine affects nAChRs expression.  These changes may contribute to the higher incidence of morbidity and mortality of those exposed to nicotine in utero through maternal smoking.  [This work was supported by NIH-MBRS S06 GM 08016-32-2 to MDG & by Kuwait University grant KU0653 to AM & MDG]