Poster Abstracts

Superfund Basic Research Program 2004 Annual Meeting: Application of Molecular Technologies for Reducing Uncertainties in Risk

Presenters

1 - Biomolecule and Hydroxyl Radical-Based Techniques for Detoxification of Hazardous Organics

Deepak Ahuja, Y.Li, L. Bachas, and D. Bhattacharyya

University of Kentucky SBRP

The presence of chloro-organics in various Superfund sites is well documented in the literature. Chlorinated organics range from chloroethylenes (such as the degreasing solvent, trichloroethylene, TCE), chlorophenols, PCBs, etc. Our research is directed towards the development of both oxidative (hydroxy radical-based) and reductive (vitamin B12 - based) platforms suitable for highly effective remediation strategies. The main difference between oxidative and reductive processes is complete reaction in the former leads to the formation of organic acids and CO2 , while for the latter results in formation of the non-chlorinated analog of the parent compound (e.g., ethylene/ethane from TCE). Our recent studies have dealt with destruction of trichloroethylene (TCE) and 2, 4, 6- trichlorophenol (TCP). The reductive pathway used the biomolecule Vitamin B12 and the electrochemical regeneration (rather than Ti citrate) of Co (III) to Co (I). This required the immobilization of Vitamin B12 molecule in a conductive polypyrrole film. On the other hand, the use of an oxidative process (such as hydroxy-radical based Fenton reaction) requires the use of Fe (II) and H2O2 to generate free radicals. Our novel approach uses potential on-site H2O2 generation (by an enzymatic process) and the use of chelate to control the reaction pathway. Chelate modified Fenton reaction is one of the techniques that can be used near neutral pH condition for in situ remediation applications. The aim of this study was to couple the H2O2 generating glucose oxidation reaction (with GOx) and the gluconic acid product as a chelate with the modified Fenton reaction. The oxidation of glucose was studied using free and immobilized enzyme glucose oxidase, and the rate of generation of hydrogen peroxide for each system was determined. Our results with TCP degradation using enzyme produced H2O2 and gluconate showed that dechlorination can indeed be achieved near neutral pH condition. This study is supported by the NIEHS-SBRP program.

 Back to top

2 - Developmental Exposure to Chlorpyrifos Elicits Sex-Selective Alterations of Serotonergic Synaptic Function in Adulthood

Justin E Aldridge, FJ Seidler and TA Slotkin

Duke University SBRP

During brain development, serotonin (5HT) provides essential neurotrophic signals and we found that gestational or neonatal exposure of rats to the commonly used insecticide chlorpyrifos (CPF) elicits immediate changes in 5HT systems. In the current study, we evaluated the effects in adulthood after CPF exposures from the neural tube stage (gestational days GD9-12) and the late gestational period (GD17-20), through postnatal (PN) neuronal differentiation and synaptogenesis (PN1-4, PN11-14), utilizing treatments below the threshold for systemic toxicity. With exposure on GD9-12, CPF elicited global elevations in 5HT1A and 5HT2 receptors and in the 5HT presynaptic transporter. The GD17-20 treatment elicited larger effects that displayed selectivity for regions with 5HT nerve terminals and that were preferential for males. Although similar receptor upregulation was seen after PN1-4 exposure, the effects were larger in regions with 5HT cell bodies; in addition, the presynaptic transporter was downregulated in the nerve terminal zones of females. The PN11-14 exposure had much smaller effects on receptors but still elicited transporter suppression with the same regional and sex selectivity. Although CPF exposure on GD17-20, PN1-4 or PN11-14 altered the ability of 5HT to modulate adenylyl cyclase, these did not correspond to the effects on 5HT receptors, suggesting an additional set of effects on proteins that transduce the 5HT signal. Our results indicate that CPF elicits long-lasting changes in 5HT receptors, the presynaptic 5HT transporter, and 5HT-mediated signal transduction after exposure in discrete developmental windows that range from the neural tube stage through synaptogenesis. These effects are likely to contribute to neurobehavioral teratology of CPF. Supported by NIH ES10387 and ES10356.

 Back to top

3 - Differential gene expression in peripheral blood lymphocytes among arsenic exposed individuals with and without arsenical skin lesions in a Bangladesh population

Maria Argos, M.G. Kibriya, F. Parvez, M. Rakib-Uz-zaman, H. Ahsan

Columbia University SBRP

We conducted microarray-based gene expression analyses, using RNA from peripheral blood lymphocytes of forty individuals selected from the Health Effects of Arsenic Longitudinal Study (HEALS), to assess the impact of arsenic exposure and arsenic-induced skin lesion status on genome-wide gene expression patterns. Three groups of participants were selected, matched on age and gender: i) 13 subjects with low arsenic exposure and without arsenical skin lesions, ii) 12 subjects with high arsenic exposure but without arsenical skin lesions, and iii) 15 subjects with existing arsenic-induced skin lesions. Gene expression levels were measured using the Affymetrix HG-U133A oligonucleotide genechip array. We examined whether arsenic exposure was associated with altered gene expression among individuals without skin lesions. Among non-smokers, a subset analysis of the high exposure (well water arsenic concentration >100 micrograms per liter) and low exposure (well water arsenic <20 micrograms per liter) groups revealed differential gene expression by arsenic exposure status. We identified 331 genes differentially expressed between the low and high exposure groups. We also examined whether genes were differentially expressed across the low exposure, high exposure and skin lesion groups and identified approximately 900 differentially expressed genes across these groups, which in hierarchical cluster analysis discriminated individuals with arsenical skin lesions from individuals without lesions. Our findings demonstrate that microarray-based gene expression analysis is a powerful method to characterize the molecular profile of arsenic exposure and arsenic-induced diseases. Genes identified from these analyses may provide insights into the underlying disease mechanisms of arsenic and represent targets for disease prevention.

 Back to top

4 - Application of the CALUX Recombinant Cell Bioassay for Detection and Relative Quantitation of Halogenated Dioxins and Related Chemicals in Environmental Matrices

David S. Baston, David J. Brown, Michael Chu, George C. Clark, Hiroshi Murata, Michael S. Denison

University of California, Davis SBRP

Halogenated aromatic hydrocarbons (HAHs), such as polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls, are a large group of highly toxic environmental chemicals that have been identified worldwide in wildlife and human tissues and in food and environmental matrices. Although detection and quantitation of these chemicals in environmental and biological matrices is critical, instrumental analysis (gas chromatography/high resolution mass spectrometry (GC/HRMS)) procedures to identify and quantitate HAHs are very costly and time-consuming. Taking advantage of the molecular mechanism of action of HAHs, we developed novel recombinant cell lines that respond to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related HAHs with the induction of firefly luciferase. The Chemically-Activated LUciferase eXpression (CALUX) cell bioassays are extremely sensitive and have been used to accurately estimate the relative potency (bioassay TEQs) of environmental, biological and food sample extracts containing complex mixtures of these chemicals. Extraction, clean-up and analysis of a variety of environmental matrices (soil, ash, exhaust gas) in double-blind validation studies revealed an excellent correlation between TEQs estimated by the CALUX bioassay and GC/HRMS, demonstrating its utility as a screening assay for the presence of bioactive/toxic HAHs in sample extracts. Quantitative analysis of these samples by CALUX suggests they contain higher concentrations of bioactive HAHs than estimated using GC/HRMS, but identity of these chemicals are unknown. The CALUX cell bioassay provides an accurate estimate of the relative amount of TCDD-like HAHs present in samples and it can be used to prescreen large numbers of samples to identify those for subsequent analysis by the more costly and time-consuming GC/HRMS procedures.

 Back to top

5 - Polymorphisms in Glutathione Related Pathway Genes and Their Association

Lynn M. Bekris, Morgan Peterson, Brian Van Yserloo, Elizabeth Rutledge, Jinko Graham, Brad McNeney, Jean Shin, Marta Janer, Marjan Zarghami, Frederick Farin, Terrance J. Kavanagh, Ake Lernmark

University of Washington SBRP

Type 1 diabetes mellitus (T1D) is an autoimmune disease involving pancreatic beta-cell destruction by immune effector cells and associated with autoantibodies to islet cell autoantigens such as GAD65, IA-2 and insulin . GSH protects against reactive oxygen species (ROS)-mediated cell injury in response to a variety of toxicants, maintains the thiol status of proteins, and is important for immune response and cytokine production. Peripheral blood GSH levels are depleted in many diseases such as coronary artery disease, Parkinson's disease and diabetes. In this investigation we hypothesized that enzymes that play a role in GSH homeostasis may be susceptibility factors for T1D including glutamate-cysteine ligase catalytic (GCLC) subunit (rate limiting for GSH biosynthesis), and the glutathione-s-transferases, mu (GSTM1) and theta (GSTT1), which conjugate GSH to various endogenous and exogenous compounds. To determine if polymorphisms in these GSH related pathway genes are associated with patient age-at-onset, gender or autoantibodies, we genotyped T1D patients (ages 0-34 years) and age-matched control subjects from Sweden. The results indicate that GCLC polymorphisms do contribute to T1D risk. The GCLC trinucleotide repeat (TNR) polymorphism was most important in T1D female patients and in patients with an earlier age-at-onset. The GCLC -129 SNP may be associated with increased GAD65 autoantibody levels in delayed age-at-onset T1D patients, and the GSTM1 and GSTT1 null genotypes have a negative association with T1D. Taken together these results suggest that GSH synthesis and GST mediated GSH conjugation play a role in T1D risk. Supported by NIH Grants 5P42ES004696, 5T32ES007032, 5P30ES007033. DK26910 and DK53004.

 Back to top

6 - The Development and Application of Microbial Molecular Biomarkers of Ecosystem Health

Nicolas Bouskill, Tim Ford

Harvard University SBRP

The upper Clark Fork River, Montana, was designated an EPA Superfund site in 1985, due to considerable metal contamination, with notably high levels of arsenic and copper from the Anaconda smelter, posing potential ecological and human health effects. As part of ongoing research into the development of microbial biomarkers as ecotoxicological tools, sediments from sites along the Clark Fork River are being evaluated for the presence of copper and arsenic detoxification genes, and for the presence of cellular stress genes. Additionally, we are attempting to quantify expression of stress gene prevalence using Real Time PCR, standardized to Pseudomonas 16S genes. Furthermore, denaturant gradient gel electrophoresis (DGGE) is being used to analyze community diversity between polluted and reference sites, providing more information about the degree of metal impact within the sites. To this end, genomic DNA is extracted from sediments and primers designed to specifically target stress genes of the Pseudomonas genome are used to amplify genes of the Ars operon (Ars R, C) the Cop operon (Cop A, B), oxidative stress systems (SOD, A, B), heat shock proteins (Hsp 32) and a bacterial metallothionein (Smt B). Current results point towards successful amplification of stress genes directly from contaminated sites, and an increasing microbial diversity within sites of correspondingly higher metal pollution. The current results represent a significant advance in using microorganisms as ecotoxicological tools.

 Back to top

7 - Combined Photolytic and Microbial Degradation of PAH

Johnnie Chamberlin

Duke University SBRP

PAH research often explores degradation in very 'non-real world' conditions. Typical studies involve bacterial isolates being grown on PAH in the absence of light. These conditions simplify research, but don't reflect the conditions found at our Superfund Site on the Elizabeth River in Norfolk, VA. In the tidal waters of this site, PAH can be found in soil containing a wide array of PAH degrading bacteria. These compounds are also found in the water or on the surface of the soil, which is exposed to hours of sunlight on an average day. My research looks into the differences between single isolate degradation of PAH and the combined effects of mixed cultures and PAH exposure to UV light. Degradation rates and end products of four isolated strains will be compared to those of mixed cultures. Microbial degradation of PAH UV degradation products and UV degradation of biodegradation products will also be discussed.

 Back to top

8 - Nickel Exposure Decreases Cellular Iron Level and Converts Cytosolic Aconitase to Iron Regulated Protein 1

Haobin Chen, Todd Davidson, Max Costa

New York University SBRP

Nickel compounds are well-established carcinogens and are known to initiate a hypoxic response in cells via the stabilization and transactivation of hypoxia inducible factor 1 alpha. This change may be the consequence of nickel's interference with the function of several Fe(II)-dependent enzymes. In this study, the effects of soluble nickel exposure on cellular iron homeostasis were investigated. Nickel treatment decreased both mitochondrial and cytosolic aconitase activity. Cytosolic aconitase was converted to iron regulatory protein 1, a form critical for the regulation of cellular iron homeostasis. Iron treatment reversed the effect of nickel on cytosolic aconitase and iron regulatory protein 1. The increased activity of iron regulatory protein 1 after nickel exposure stabilized and increased transferring receptor mRNA and antagonized the translation of ferritin light chain protein. The decrease of aconitase activity after nickel treatment reflected neither direct interference with aconitase function nor obstruction of [4Fe-4S] cluster reconstitution by nickel. Soluble nickel exposure decreased total cellular iron by about 40%, a decrease that likely caused the observed decrease in aconitase activity and the increase of iron regulatory protein 1 activity. This disturbance of cellular iron homeostasis by nickel may have a great impact on the ability of the cell to regulate a variety of cell functions.

 Back to top

9 - Association between Low-dose Arsenic Exposure from Drinking Water and High Blood Pressure in Bangladesh

Yu Chen, P. Factor-Litvak, F. Parvez, J.H. Graziano, G. Howe, H. Ahsan

Columbia University SBRP

We evaluated the effect of arsenic exposure from drinking water on high blood pressure in a cross-sectional study using baseline data from 11,458 participants recruited in the Health Effects of Arsenic Longitudinal Study (HEALS) in Bangladesh. Blood pressure was measured with an automatic sphygmomanometer by a trained physician according to recommended guidelines. Arsenic exposure was estimated from well water concentration. Logistic regression modeling of clustered data using generalized estimating equations (GEE) was conducted to estimate prevalence odds ratios (PORs) for systolic hypertension (SBP ≥ 140 mmHg), diastolic hypertension (DBP ≥ 90 mmHg), general hypertension (SBP ≥ 140 mmHg and/or DBP ≥ 90 mmHg), and high pulse pressure (PP ≥ 55 mmHg) by levels of arsenic exposure. In contrast to those in the lowest quintile of well arsenic (<7.0 μg/L, mean 2.4 μg/L), participants with higher well arsenic (7.3-91.0 μg/L, mean 42.7 μg/L; 92.0+ μg/L, mean 211.2 μg/L) experienced greater risks of systolic hypertension (POR=1.38, 95% CI 1.12-1.69; POR=1.30, 95% CI 1.05-1.59, respectively) and high pulse pressure (POR=1.31, 95% CI 1.09-1.56; POR=1.21, 95% CI 1.01-1.44, respectively). Additionally, data indicated marked stronger associations in those with higher BMI and/or poor nutritional intakes of Vitamin B12, B6, B2, and folate. PORs were similar when arsenic exposure was defined by a cumulative arsenic index which accounts for water consumption and well use history. No significant associations were found when urinary arsenic was used as measure of exposure. The findings suggest potential harmful cardiovascular effects of arsenic exposure <100 μg/L or higher.

 Back to top

10 - Oxidation of 2'-deoxyguanosine and 8-Oxo-2'-deoxyguanosine by Dimethyldioxirane

Diana E. Degen, Ball, L.M., Gold, A., Sangaiah, R., Li, Yutai

University of North Carolina SBRP

Oxidative damage to DNA can lead to cancer, aging, and neurological disorders. Guanine is the most susceptible to oxidation out of the four DNA bases. The oxidation product of 2'-deoxyguanosine (2'-dG), 8-oxo-2'-deoxyguanosine (8-oxo-2'-dG), is even more susceptible to oxidation than 2'-dG, and for this reason, it is imperative to study its oxidation products as well. Therefore, to help us further our knowledge of how oxidation contributes to illness and disease, it is important to study the oxidation process and products of 2'-deoxyguanosine. Oxidations of 2'-deoxyguanosine and 8-oxo-2'- deoxyguanosine were conducted using the powerful chemical oxidant Dimethyldioxirane (DMDO). Oxidation products were then characterized with the use of HPLC, proton and multidimensional heteronuclear NMR, and electrospray MS in the positive ion mode. The major product fraction was formed from epoxidation of the C4-C5 double bond followed by solvolysis. Hence, in the reaction of DMDO with 2'-deoxyguanosine, epoxidation followed by hydrolysis formed 4,5-dihydro-4,5-dihydroxy-2'-deoxyguanosine. Accordingly, when DMDO reacted with 8-oxo-2'-deoxyguanosine, epoxidation followed by hydrolysis resulted in the formation of 4,5-dihydroy-4,5-dihydroxy-8-oxo-2'-deoxyguanosine and epoxidation followed by methanolysis resulted in the formation of 4,5-dihydro-4-hydroxy-5-methoxy-8-oxo-2'- deoxyguanosine. The latter was further verified by conducting oxidations in perdeuterated methanol. This research concludes that DMDO, which has an oxidation mechanism analogous to the mixed-function oxidases, generates products distinct from those arising from radical chemistry.

 Back to top

11 - Reductive Dechlorination of the Vinyl Chloride Surrogate Chlorofluoroethene in TCE-Contaminated Groundwater

Elliot Ennis, Ralph Reed, Mark Dolan, Lewis Semprini, Jonathan Istok, Jennifer Field

Oregon Health and Science University SBRP

Trichloroethene (TCE) was used extensively as an industrial degreasing agent and improper disposal and leaks have led to groundwater contamination. At many sites, microbial transformation of TCE results in the accumulation of vinyl chloride (VC), a known carcinogen and neurotoxin. Although qualitative evidence for the transformation of VC to ethene can be obtained by a number of field methods, quantitative tools are needed to determine the in situ rates of VC transformation to ethene in contaminated groundwater. The rate of reductive dechlorination of chlorofluoroethene (CFE) to form fluoroethene (FE) was determined in push-pull tests conducted at a TCE-contaminated field site and in laboratory microcosms prepared with aquifer material from the site. Rates for VC transformation to ethene and the sum of CFE isomer transformation to FE were within a factor of two for the microcosms systems, which indicated that CFE can be used to predict the in situ transformation rate of VC to ethene. The single well push-pull tests consisted of injecting E-/Z-CFE and monitoring for the formation of FE over a period of 80 days. Reductive dechlorination of CFE to FE was observed in four out of six test wells with total CFE disappearance rates ranging from 0.007 to 0.11 day-1. This work was supported by grant number ES10338.

 Back to top

12 - Quantitation of Selected Microbial Populations in Chloroethene-Contaminated Groundwater

Whitney J. Fraser, Hannah-Malia A. Viernes, Russell P. Herwig

University of Washington SBRP

Chlorinated ethenes are a human health hazard, and are currently among the most common groundwater pollutants found in the United States. In anoxic groundwater environments, highly chlorinated ethenes are generally slow to degrade. However, there exist anaerobic bacteria that can progressively remove chlorines during respiratory processes in which the chlorinated ethene is used as a terminal electron acceptor. These halorespiring bacteria are a diverse group of organisms important for bioremediating recalcitrant toxic compounds, particularly chlorinated ethenes. The purpose of this project is to construct a quantitative profile of halorespiring bacterial taxa and, further, to investigate whether the profile or any subset of thereof correlates with the profile of a contaminant plume as delineated through concomitant sampling. Samples were collected from two contaminated groundwater sites in Oregon. Specific prokaryotic taxa in the groundwater will be quantified by applying a 5' nuclease assay (also known as quantitative or 'real-time' PCR) to DNA extracted from 30 mL water samples. The ultimate use of this profiling technique could be as a diagnostic aid in characterizing toxic sites, as a tool to compare the effectiveness of different remediation methods, and as a source of basic knowledge about the ecology of dechlorinating bacterial communities.

 Back to top

13 - Arsenic Oxidation by Aerobic Chemoautotrophic Microorganisms from Soil and Sediments

Elizabeth Garcia-Dominguez, E. D. Rhine, A. Paschal, and L. L. Young

New York University SBRP

Microorganisms able to oxidize arsenite have been isolated from extreme environments under aerobic conditions and may play an essential role in arsenic biogeochemical cycling. Three aerobic Gram-negative strains were successfully isolated from enrichments cultures that were able to rapidly oxidize 10 mM of As(III) to As(V) under aerobic conditions using CO2/HCO3= as the carbon source. Based on 16S rDNA sequence analysis, strain OL-1, from Onondaga Lake sediment (NY), is most closely related to an Ancylobacter, strain S-1 from an arsenic contaminated soil (NJ) is 99% related to Thiobacillus, and strain CL-3, from an artificial colleting lagoon located in a oil refinery facility (Venezuela), is 98% related to the genus Hydrogenophaga. Cell growth of the isolates showed preference for sulfur species (sulfur, sulfide and thiosulfate) except for CL-3 which also can grow on ammonium and NO2. The RuBisCO Type I (cbbL) gene was positively amplified and sequenced in strain CL-3, and the Type II (cbbM) gene was positively amplified in strains OL-1 and S-1. Kinetic studies comparing the rate of oxidation for each electron donor showed a rapid transformation of As(III). These results confirm the ability of these strains to autotrophically grow while oxidizing As(III). Since As(III) is more mobile and As(V) can be readily sorbed to clays and minerals, arsenic oxidation could potentially retard the movement of arsenic in the environment by making it less bioavailable. Studying the processes by which microorganisms aid in the biogeochemical cycling of arsenic can help in developing more efficient methods for remediation.

 Back to top

14 - Leaching from Solid Residuals of Arsenic Treatment under Landfill Conditions

Amlan Ghosh, Muhammed Mukiibi, Eduardo Saez, Wendell Ela

University of Arizona SBRP

Most of the estimated 8 million pounds of arsenic residuals that would be generated annually through compliance with the new arsenic MCL, pass the TCLP and may be disposed in non-hazardous landfills. In this research, arsenic residuals (Activated Alumina (AA), Granular Ferric Hydroxide (GFH) and Amorphous Ferric Hydroxide (AFH) sludges) were subjected to TCLP and WET (Waste Extraction Test) leaching tests and actual landfill leachates. The results indicate the standard testing methods significantly underestimate the arsenic mobilization from the residuals, because critical physical and chemical dissimilarities exist between the test and landfill conditions. When arsenic residuals were subjected to reducing, landfill conditions inside long-term, continuous flow columns, microbes reduced Fe(III) to Fe(II) and As(V) to As(III). Arsenic was associated largely with particulate-phases, rather than as a dissolved-phase. Sorbent (iron) leaching may be faster than sorbate (arsenic) leaching, leading to a sudden spike in arsenic release when the sorbent is exhausted. Under long residence times in landfills, aging and re-mineralization of solid residuals may alter their arsenic retention. AFH, the residual generated when arsenic is removed from ion exchange and membrane process brine streams, is expected to age to goethite and hematite. At low As: Fe ratios, this expected trend occurs, whereas at high As: Fe ratios a distinct mineral, pharmocosiderite (hydrated sodium iron arsenate hydroxide), evolves. The aging rate and distribution of crystallized iron phases is a function of pH and temperature, as well as arsenic concentration.

 Back to top

15 - Interactions of Complex Chemical Mixtures from Contaminated Sediments at a Superfund Site

Annika Gillespie, T.D., McDonald, T.J., He, L-Y., Zhou, G-D., and Donnelly, K.C.

Texas A&M University SBRP

Because of multiple pathways of exposure and potential interactions of chemical mixtures, it is difficult to develop an accurate risk assessment for chemically contaminated sites. These include the Superfund sites. Risk characterization is the initial step towards ranking sites and evaluating appropriate remedial procedures. Chemical mixture interactions may alter transport across a membrane, induction of metabolizing enzymes or binding with critical macromolecules. The current study investigated potential interactions of mixtures of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) extracted from sediments at a Superfund site. Sediment samples were collected from a reference station and five locations along a contaminated river in the Pacific Northwest. Dried sediments were extracted and analyzed for PAHs, semi-volatile organics and PCBs. Sediment extracts were tested in vitro using microbial genotoxicity bioassays and in vivo using female ICR mice. The samples were either negative or weakly positive in the microbial bioassays. Animal studies indicated that the genotoxicity of sediment extracts was more strongly correlated with PAH content than with the concentration of PCBs. When the sediment extracts were co-administered with benzo(a)pyrene (BAP) to mice, the complex mixtures appeared to inhibit the formation of BAP-DNA adducts. Application of increasing concentrations of sediment extract reduced the levels of DNA adducts attributed to the BAP-diol epoxide (Spot #4). Chemical analysis identified high molecular weight PCBs as the major contaminant. Total PCB concentrations were generally at least 4-5 times greater than total PAH concentrations. These data suggest that chemical interactions reduced the potential genotoxicity of the complex mixtures extracted from sediments.

 Back to top

16 - Association between inorganic arsenic exposure and increased vibrotactile threshold in Bangladesh

Danella Hafeman, J.H. Graziano, E.D. Louis, A. Siddique, Z. Cheng, V.N. Slavkovich, H. Ahsan

Columbia University SBRP

This pilot study examined the association between chronic arsenic exposure and peripheral neuropathy in Araihazar, Bangladesh, where the population has been chronically exposed to varying levels of arsenic through naturally contaminated tube well drinking water. We conducted a cross-sectional study of 137 subjects derived from a larger cohort. Exposure measures included individual water arsenic concentration, cumulative arsenic index (water arsenic multiplied by years exposed), and urinary arsenic concentration taken at both the time of outcome measurement (2003) and two years prior to examination (2001). The primary outcome measurement was vibrotactile threshold of the index finger and great toe, as measured by a vibration sensitivity tester (Vibratron II); data on a number of secondary outcomes (proprioception, grip strength, reflexes, and tapping speed) was also collected. The subjects were exposed to a wide range of arsenic concentrations; the mean water arsenic concentration was 115 μg/L, with a range of 5 to 743 μg/L. Arsenic exposure was associated with elevated toe vibration threshold (TVT) but not with elevated index vibration threshold (IVT) or changes in secondary outcomes. Specifically, linear regression analyses showed that cumulative arsenic index and urinary arsenic (2001) were both statistically significant predictors of TVT (p=0.02 and p=0.009, respectively) after adjustment for age and gender. In conclusion, increased arsenic exposure, as measured by both cumulative and urinary measures, was associated with evidence of subclinical sensory neuropathy. We plan to conduct additional studies to examine the dose-response relationship.

 Back to top

17 - Aromatic Hydrocarbon Neurotoxicity: Assessment of CNS Protein Vulnerability In Vitro

Seyed BS Hashemi, AP Reddy, M Lasarev, S Nagalla, PS Spencer, Mohammad I. Sabri

Oregon Health and Science University SBRP

Rodents treated systemically with the protein-reactant hydrocarbon solvent metabolite 1,2-diacetylbenzene (1,2-DAB), but not with its non-protein-reactant isomer 1,3-DAB, develop nerve fiber damage (neurofilamentous axonal swellings) in the central nervous system (CNS). We examined CNS protein targets of DAB isomers by 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Spinal cord slices were incubated with 5-20 mM of either DAB isomer or 2% acetone-based vehicle for 30 min at 37 0C, solubilized in rehydration buffer and centrifuged (10,000 x g for 1 hr). Solubilized protein aliquots (~350 mg) were focused on 24-cm immobilized pH gradient strips (pH 4-7) and then separated on 5-20 % sodium dodecyl sulfate polyacrylamide gels. 1,3-DAB had no significant effect on the number of proteins (489 and 488 proteins at 5 and 20 mM, respectively) relative to vehicle-treated controls (508). By contrast, equivalent concentrations of 1,2-DAB reduced the number of detectable proteins (392 and 237, respectively). Proteins of high molecular weight (MW) and high isoelectric point (pI) were especially vulnerable to 1,2-DAB (~ 40% & ~90% reduction at 5mM & 20mM, respectively). Low-MW and low-pI proteins were least affected by 1,2-DAB. Lysine-rich neurofilament (NF) subunit (NF-M) was more susceptible than lysine-poor NF subunit (NF-L) as shown by LC/MS/MS using a hybrid quadruple time-of-flight mass spectrometer. These data suggest that neuroprotein vulnerability to the neurotoxic g-diketone 1,2-DAB is directly related to lysine content, molecular mass and isoelectric point. [Supported by NIEHS grants 10338 & 011384 and the Oregon Workers Benefit Fund]

 Back to top

18 - Natural Plant Populations on an Aged Petroleum Refinery Landfarm: Phytotoxicity, Stabilization, and Degradation of Contaminants

Heather F. Henry, Jodi. R. Shann

University of Cincinnati SBRP

Allowing natural ecological restoration on aged contaminated sites is a sustainable end-use strategy that provides greenspace for wildlife and may continually degrade or stabilize remaining contaminants. Bioassays (Lactuca sativa) can gauge site phytotoxicity of soil and, therefore, the necessity for a clean soil cap. However, early successional wild species may be more informative than plant species typically used for bioassays. In this study, soil samples from a highly contaminated area of a petroleum refinery landfarm were tested for phytotoxicity to a local plant species (Solidago canadensis, Canadian goldenrod) beginning to establish on the site. There was a slight negative response in the wild population bioassay; however, there was also evidence that resistant individuals existed. Total petroleum hydrocarbons and sequential extraction of metals were used to determine the contaminant content in the test soil and across the 5.5 acre site. Given low bioavailability of soil contaminants, it was concluded a clean soil cap was not necessary. As validation for the bioassay assessment, establishment of goldenrod onsite was monitored for three years. Initially found in only two perimeter plots, goldenrod became established throughout the entire site within three years. Analysis of petroleum and metal contaminants after three years indicated TPH and PAHs had reduced by over 50% - metals (total and within sequential fractions), did not change nor was there significant uptake into plant tissue. This study demonstrates the potential for stabilization and degradation of contaminants by natural populations establishing on contaminated sites.

 Back to top

19 - Assessing the Human Health Threat from CCA-treated Mulch

Crystal M. S. High, Daniel B. Oerther

University of Cincinnati SBRP

Historically, CCA (chromated copper arsenate) was the most widely used inorganic wood preservative. Because arsenic and chromium are known carcinogens, it is important to determine the fate of CCA-treated lumber in the environment. Prior research indicates that CCA-treated lumber has been incorporated into mulch feed stock. The objective of this study was to assess the threat to human health from CCA-treated lumber in mulch. Forty samples of commercial mulch were purchased in the Cincinnati, OH metropolitan area. Metals were extracted according to TCLP (toxicity characteristic leaching procedure) and analyzed by ICP/MS. Thirteen mulch samples had Cr concentrations greater than 5ppm, and six mulch samples had As concentrations ranging from 0.4 - 2.2 ppm. No correlation was observed between the levels of Cr, Cu, and As recovered by TCLP. The bioavailability and bioaccumulation of Cr, Cu, and As released from mulch is currently being assessed in plant studies. Green ice lettuce, better boy tomatoes, and little finger carrots have been planted in sandy loam soil with a two inch ground cover of CCA-contaminated mulch. It is expected that Cr, Cu, and As will leach from the mulch and accumulate within the plants. The results of this study should provide an initial survey of the potential threat to human health from CCA treated lumber recycled as part of mulch feed stocks.

 Back to top

20 - The use of a passive, integrative, in situ sampler to assess the influence of seasonal and episodic events on the distribution of bioavailable manganese (Mn) and trace metals in the lower Willamette River, Portland Harbor Superfund site, OR

Wendy E. Hillwalker, Oraphin Krissanakriangkrai, Robert Grove, Kim A. Anderson

Oregon Health and Science University SBRP

An 18 mile stretch of the lower Willamette River, OR, was investigated for the distribution of bioavailable trace metals from 2001 to 2004. The areas assessed included the Portland Harbor Superfund site (RM 3.5 to 9.5) and the McCormick and Baxter Superfund site (RM 7). Designated Superfund status in 2000, Portland Harbor is heavily industrialized, contaminated with metals, polychlorinated biphenyls (PCBs), dioxins/furans, pesticides, and polyaromatic hydrocarbons (PAHs). Alternative sampling methods, including in situ sampling devices and aquatic organism collection, were used to determine the influence of seasonal and episodic events on the distribution of bioavailable manganese (Mn), copper (Cu), chromium (Cr) and zinc (Zn) in the Portland Harbor and McCormick and Baxter Superfund sites. Diffusive gradients in thin film (DGT) is an in situ, time integrated, passive sampling device containing a chelex resin with high selectivity for labile, divalent cations. DGT were deployed within the main river channel near urban, industrial and Superfund land uses during a range of river conditions from 2001 to 2004. Two species of predatory fish were collected along the 18 mile stretch of the lower Willamette River in 2000, and crayfish were collected at the same locations as DGT deployment in 2003. Preliminary data indicates that bioavailable trace metal concentrations were influenced by episodic events, such as terrestrial run-off and dilution, seasonal variations in river flow and precipitation, as well as land use factors influencing metal source to the river. Trace metal bioaccumulation by organisms varied relative to organism- and site-specific factors.

 Back to top

21 - The impact of cadmium and environmental factors on naphthalene removal by a multi-species biofilm

Peng Jin, Paul Bishop

University of Cincinnati SBRP

Biofilm technology is being introduced into bioremediation of contaminated soils and underground water, in the form of biofilm barriers. However, the low availability of PAHs and toxicity of the frequently accompanying heavy metals may limit its application. The objective of this study was to investigate the mass transfer and distribution of cadmium into the biofilm matrix and the impact caused by it. The influences of pH value, presence of an alternative substrate and increased dissolved oxygen were examined. As the pH value increased, cadmium sorption in the biofilm increased and naphthalene removal decreased. Ten mg/L of cadmium was enough to show a significant impact on biofilm when the pH was above 7.5. The cadmium minimum inhibition capacity was determined to be 5.0 μgCd/mgVS. Acetate, added as an alternative substrate, competed with naphthalene and did not demonstrate the ability to reduce cadmium toxicity. Hydrogen peroxide, added to supplement the dissolved oxygen, accelerated the cadmium uptake/efflux cycle, making the biofilm more vulnerable to cadmium attack. Cadmium was shown to transfer faster than naphthalene into biofilm, and the removal of naphthalene in the presence of cadmium was determined to be solely due to sorption.

 Back to top

22 - Pore Water and Sediment Profiles of Arsenic From a Contaminated Stream and Lake

Alison R. Keimowitz, Yan Zheng, Steven N. Chillrud, Hun Bok Jung, Yi He, Brian J. Mailloux, Martin Stute, H. James Simpson

Columbia University SBRP

The Vineland Chemical Company (Vineland, NJ) manufactured arsenical herbicides for ~50 years during which time groundwater and soils beneath the plant became heavily contaminated with arsenic. Groundwater discharge to the Blackwater Branch, a small stream adjacent to this Superfund site, allowed significant As transport downstream to the Maurice River and Union Lake. Sediment cores obtained from the Blackwater Branch and Union Lake in July 2004 were sectioned within 2 hours in a nitrogen atmosphere and pore waters were examined for arsenic speciation; iron speciation; and total concentrations of arsenic, iron, and manganese. Sediments were extracted with a 1 M phosphate buffer under anoxic conditions for 24 hours followed by 1.2 M HCl for 1 hour. The phosphate extractant was examined for arsenic speciation and both extractant's were examined for total concentrations of arsenic, iron, and manganese. In the Blackwater Branch core, pore waters contain <1,200 ppb arsenic, 68-100% As(III), and sediments contain <250 ppm arsenic. In Union Lake, pore waters contain <2,000 ppb arsenic, 48-96% As(III), and sediments contain <1,250 ppm As. Preliminary analysis of sediment and pore water profiles indicate that in the stream, sediments are a net sink of arsenic introduced via groundwater discharge. These data may help guide remediation decisions at this site (e.g. whether or not to dredge the Blackwater Branch and/or Union Lake), as well as improve a general understanding of groundwater arsenic contamination to surface water bodies.

 Back to top

23 - Inter-individual variability in benzene metabolites

Sungkyoon Kim, R Vermeulen, S Waidyanatha, Q Lan, MT Smith, GL Li, L Zhang, M Shen, S Yin, N Rothman, SM Rappaport

University of North Carolina SBRP

The deleterious effects of benzene exposure are thought to be caused by the production of cytotoxic and genotoxic metabolites. Since metabolism varies among individuals, we investigated interindividual variability in the production of benzene metabolites among 116 workers exposed to benzene in China (mean exposure = 4.8 ppm; 10th-90th percentiles 0.40-10 ppm). Each worker had repeated measurements of both personal airborne exposure to benzene and toluene and urinary benzene metabolites (tt-muconic acid, S-phenylmercapturic acid, phenol, catechol, and hydroquinone), as well as of unmetabolized benzene in urine. We used mixed effects statistical models that incorporated random slope and intercept terms for each worker to evaluate interindividual variability. In this context, the random slope explains the subject-specific relationship between each metabolite (or unmetabolized benzene) and benzene exposure, while the random intercept relates to the background metabolite level for each subject (due to the presence of non-benzene sources of the metabolites, particularly the phenolic compounds). Significant interindividual variability was detected for each benzene metabolite and for unmetabolized benzene in urine. Fixed effects were included in the models to investigate the influence of potential covariates on benzene metabolite levels, notably co-exposure to toluene, lifestyle factors, and smoking. Several of these covariates were significantly associated with the production of benzene metabolites. * Supported by grant P42ES05948 from NIEHS/NIH

 Back to top

24 - Monitoring Bioaugmenation with Single-Well Push-Pull Tests in Sediment Systems Contaminated with Trichloroethene

Jae-Hyuk Lee, Ralph Reed, Jennifer Fiel, Mark Dolan, Jonathan Istok

Oregon Health and Science University SBRP

Bioaugmentation, the introduction of microorganisms to degrade a target compound in a contaminated medium, is one approach that has been investigated in an effort to accelerate the reductive dechlorination activity in situ. The effectiveness of bioaugmentation in bench scale and field scale studies depends on (1) whether the dechlorinating activity in situ is due to indigenous or bioaugmented strains and (2) the extent of penetration of injected culture around the injection wells. A lab-scale, single-well, push-pull test for bioaugmentation was developed to monitor reductive dechlorination of trichloroethene (TCE) and trichlorofluoroethene (TCFE) using a dechlorinating culture. The active culture was added to a physical aquifer model (PAM) to initiate reductive dechlorination of TCE and TCFE. TCE transformed to cis-dichloroethene (c-DCE), vinyl chloride (VC), and ethene, while TCFE was reductively degraded to cis-dichlorofluoroethene (c-DCFE), trans-dichlorofluorethene (t-DCFE), 1,1-chlorofluoroethene (1,1-CFE), 1,2-trans-fluoroethene (t-CFE) and fluoroethene (FE) during the 2-week test. TCE (13.0 to 46.0 mg/L) was completely degraded to ethane in less than 14 days and TCFE (15.0 mg/L) was also completely transformed to FE in 14 days. Similar transformation rates and time courses for TCE degradation and TCFE degradation were observed during the test and no transformation products of TCE and TCFE were observed for an unbioaugmented control PAM. Dehalococcoides ethenogenes was detected in the source culture and in the bioaugmented PAM but not in the control PAM. TCE and TCFE dechlorinating activity was observed across all sampling ports of the PAM, indicating the complete penetration of the culture into the bioaugmented PAM. The study shows that bioaugmentation was effective in a laboratory push-pull test. It also yields information on transformation rates and the extent of culture penetration into aquifer sediment.

 Back to top

25 - Different Ratios of Omega 6 and Omega 3 Fatty Acids Can Mediate PCB Toxicity in Endothelial Cells

Wang Lei, Gudrun Reiterer, Michal Toborek, Bernhard Hennig

University of Kentucky SBRP

The vascular endothelium is exposed to blood components including environmental contaminants and is therefore vulnerable to chemical insults. We have previously shown that exposure to coplanar PCBs can cause endothelial cell activation by inducing oxidative stress and pro-inflammatory signaling pathways. We hypothesize that dietary components such as fatty acids can modify PCB-induced cytotoxicity. Our previous studies indicate that linoleic acid, a major omega-6 unsaturated fatty acid in the American diet, can potentiate vascular endothelial cell dysfunction mediated by PCB 77. Because of the known anti-inflammatory properties of omega-3 fatty acids, we investigated the effects of different ratios of omega-6 to omega-3 fatty acids on PCB-induced endothelial cell activation. Cultured endothelial cells were pretreated with different ratios of linoleic to linolenic acids or with these fatty acids alone, followed by exposure to PCB 77. Compared to control cultures or linoleic acid-treated cells alone, supplementation with linolenic acid significantly protected against PCB 77-mediated induction of oxidative stress. A similar pattern of protective effects was found by measuring NF-kappaB DNA binding activity, which was down-regulated with a relative increase in omega-3 to omega-6 fatty acids. Omega-3 fatty acid protection against PCB toxicity was also observed when studying VCAM-1 expression induced by PCB 77 and/or linoleic acid. Endothelial cell activation by PCB 77 was reduced most markedly with a linoleic to linolenic acid ratio of 1:3. These studies suggest that dietary fats can selectively modulate vascular cytotoxicity mediated by persistent organic pollutants such as PCBs.

 Back to top

26 - Exposure Assessment in Contaminated Environments

Rebecca Lingenfelter, Leslie Cizmas, Ziad Naufal, Christine Naspinski, Ling-Yu He, Guo-Dong Zhou, Thomas McDonald, Guy Denoux, Robin Autenrieth, Arif Mekhtiev, Arif Islamzadeh, Kirby Donnelly

Texas A&M University SBRP

Humans are frequently exposed to complex mixtures of polycyclic aromatic hydrocarbons and other compounds. These include exposures due to chemically contaminated, or Superfund, sites, as well as exposures to combustion by-products. Human populations in contact with these chemicals may experience adverse health effects if the exposures are of a sufficient dose and duration. However, limited information is available to understand the relationship between environmental concentrations and absorbed dose. Studies have been conducted at a contaminated and control site in Azerbaijan to investigate the relationship between environmental concentrations and biomarkers of exposure. Data have been collected to measure environmental concentrations, and biomarkers of exposure in human populations. The biomarkers being investigated include blood plasma concentrations of PAHs, and DNA adduct formation in lymphocytes. The results indicate that concentrations of total polycyclic aromatic hydrocarbons (tPAHs) range from 2,300 ng/m2 for a window swipe to 40,400 ng/m2 for a floor swipe sample. The model carcinogen, benzo(a)pyrene, was detected in house dust at concentrations ranging from 178 ng/m2 to 483 ng/m2. Analysis of human blood plasma of all exposed individuals for PAH residues found mean total PAH levels of 303 ng/ml blood and mean carcinogenic PAH levels of 5.6 ng/ml blood. DNA adducts were significantly higher in the total exposed population than in the total control population. However adducts in lymphocytes from men in the control region were comparable to those in men from the exposed region. Overall, the results indicate there is a correlation between PAHs in environmental samples and PAH adducts in an exposed population.

 Back to top

27 - PPARgamma and PPARalpha Reduce PCB-Induced Inflammation in Endothelial Cells

Zuzana Majkova, Gudrun Reiterer, Ramadass P., Michal Toborek, Hongxia Guo, Bernhard Hennig

University of Kentucky SBRP

PCBs are widespread environmental contaminants which can lead to atherogenic events by inducing a vascular inflammatory response. To explore the mechanism of the pro-atherogenic properties of PCBs we determined the interaction of PCBs with the anti-inflammatory transcription factors peroxisome proliferator-activated receptors (PPARs) alpha and gamma. In the current study we demonstrate that PPARs can reduce the PCB-induced inflammatory process. Porcine primary vascular endothelial cells were exposed to the coplanar PCB 77, and the non-coplanar PCB 153. Both PCBs significantly induced inflammatory transcription factors, such as nuclear factor kappa-B (NF-kappaB) and signal transducer and activator of transcription-3 (STAT-3). Pretreatment of cells with a PPARalpha agonist (fenofibrate) or PPARgamma agonist (thiazolidinedione) prevented the PCB-mediated DNA binding activity of these transcription factors. In addition, PPAR agonists protected against the PCB 77 and PCB 153-induced mRNA expression of downstream inflammatory genes, such as cycloxygenase-2 (COX-2) and interleukin-6 (IL-6). Because the proinflammatory effect of PCBs could be mediated by an interference of PCBs with PPARs, we furthermore investigated the effect of PCBs on PPAR activity. Endothelial cell exposure to PCB 77 potently inhibited PPAR binding to the PPAR response element (PPRE). PPAR transactivation was also significantly reduced by all PCBs tested as determined by luciferase activity of MCF-7 cells stably transfected with a PPRE-driven luciferase. These findings suggest that the inflammation induced by PCBs is, at least in part, due to dysfunctional PPAR signaling. Our data also demonstrate that PPAR activation by synthetic agonists can significantly reduce PCB-induced proatherogenic signaling pathways.

 Back to top

28 - The Plasma Membrane Microdomains Caveolae are Involved in Toxicity of Polychlorinated Biphenyls

Zuzana Majkova, Eric Smart, Alan Daugherty, Michal Toborek, and Bernhard Hennig

University of Kentucky SBRP

Caveolae are membrane microdomains enriched in cholesterol, sphingomyelin and numerous proteins involved in cellular signaling. They are especially abundant in endothelial cells. Many proteins involved in activation of the endothelium reside in caveolae or are scaffolded by caveolin, a major structural component and marker of caveolae. Caveolin-1 knock-out mice are protected against development of atherosclerosis. PCBs are persistent lipophilic environmental contaminants that can contribute to vascular diseases. We hypothesize that caveolae signaling is critical in PCB cytotoxicity. Treatment with coplanar PCB 77 but not non-coplanar PCB 153 increases gene expression of caveolin-1 and annexin-2 in primary endothelial cells. Interestingly, exposure to PCB 77, but not PCB 153, also upregulated cyclooxygenase-2, a pro-inflammatory enzyme partially localized in caveolae. Mouse RAW macrophages, that naturally do not express caveolin-1 protein, were compared to the RAW cells stably transfected with caveolin-1. PCB 77 treatment increased DNA-binding activity of the transcription factor NF-kappaB in cells transfected with caveolin-1 compared to RAW. We further measured the oxidative stress caused by PCB 77 in both models. The DCF fluorescence was increased markedly after 4 h of PCB 77 treatment only in the presence of caveolin-1. Preliminary data demonstrate that a PCB 77-induced inflammatory response (e.g., gene expression of VCAM-1) is reduced in aorta of mice lacking the caveolin-1 gene as compared to wild-type mice. These data suggest that caveolae are involved in PCBs toxicity and also that the lack of caveolin-1 can be protective against toxicity of PCBs.

 Back to top

29 - Reactive Iron Oxide Based Adsorbent Media for Removing Arsenic from Potable Water

Dhananjay Mishra, James Farrell

University of Arizona SBRP

This research investigates the effectiveness of granular magnetite (Fe3O4) and wustite (FeO) as reactive adsorbent media for removing arsenic from potable water supplies. Past work studying arsenic adsorption to iron oxides has shown that freshly precipitated oxides are more effective adsorbents than aged materials. Therefore, this research uses a reactive adsorbent that continuously generates iron oxide adsorption sites for arsenic removal. Magnetite and wustite were selected as the reactive media for oxide generation because of their slow oxidation rates compared to that for zerovalent iron (ZVI). When iron oxides are generated much faster than they are consumed by adsorbed arsenic compounds, the excess oxides lose porosity and surface area as they age, and contribute to mass transfer limitations for arsenic removal. The planned use of the adsorbent media is in packed-bed filters in both large and small-scale treatment systems, as well as a point of use filters suitable for an individual well-head or faucet. No regeneration of the adsorbent media is planned, and disposal of the spent media is expected to be as a non-toxic waste.

 Back to top

30 - Effect of Acetaminophen on Cytosolic and Mitochondrial Glutathione in the Livers of Wild-Type, GCLM-Heterozygous, and GCLM-Null Mice

Isaac Mohar, Lisa McConnachie, Carolina Fernandez, Terrance Kavanagh

University of Washington SBRP

Acetaminophen (APAP) is metabolized by CYP450s to N-acetyl-p-benzoquinoneimine, a reactive molecule, which is detoxified via glutathione (GSH) conjugation. APAP overdose can cause GSH depletion, which is correlated with the degree of observed APAP-induced liver injury. Mitochondrial GSH (mtGSH) status is hypothesized to play a pivotal role in this process. GSH synthesis is in part controlled by glutamate cysteine ligase (GCL), a heterodimer of catalytic (GCLC) and modulatory (GCLM) subunits. In order to investigate the hypothesis that mtGSH is preferentially maintained under conditions of chronic GSH depletion and acute oxidative stress, we utilized our GCLM-null mouse model. GCLM +/+, +/-, and -/- mice (n=7 ea.) were fasted for 12-hours followed by i.p. injection of saline or APAP (300 mg/kg). Mice were euthanized 6 hours post-treatment and hepatic mitochondria isolated. Analysis of mean cytosolic GSH (ctGSH) (nmol GSH/mg wet liver) in control mice demonstrated -/- mice to contain 18% of +/+ mice. Mean ctGSH was observed to decrease by 52 and 60% upon APAP treatment of +/+ and -/- mice, respectively. Mean mtGSH in control -/- mice was not statistically different from +/+ mice. A decrease in mean mtGSH upon APAP treatment was only observed in -/- mice (68% decrease). However, as a percent of combined (ct+mt) GSH, mtGSH of -/- mice was observed to be 67 and 200% greater than mtGSH of APAP-treated and control +/+ mice, respectively. APAP treatment increased the mean percentage of GSH in mitochondria of +/+ mice by 55%. Measures of +/- mice were not statistically different from +/+ mice. These results demonstrate that under conditions of chronic GSH depletion, as occurs in -/- mice, or acute oxidative stress, as induced by APAP, mtGSH is not depleted to the same degree as ctGSH. These results support the hypothesis that mtGSH is preferentially maintained even as ctGSH is being depleted. Supported by NIH grants 1P42ES04696, 1R01ES10849, 1T32ES07032, and 1P30ES07033.

 Back to top

31 - Altered Fungal Community Structure in Forest Soils Impacted by Chromium Contamination

Sabrina R. Mueller, Brian Kinkle

University of Cincinnati SBRP

Predicting bioremediation effectiveness for sites containing both metal and organic pollutants is limited by our knowledge of short and long-term changes within metal-impacted soil microbial communities. Metals do not undergo microbial degradation, but rather persist and serve as long-term sources of stress. Previous research characterizing metal-impacted soils has focused mainly on bacterial diversity. Nevertheless, it is commonly believed that fungi are more metal-tolerant than bacteria, based on broad-scale activity measurements. The main goal of this project is to determine if fungal communities can withstand and recover from metal contamination. A microcosm study was conducted to measure the effects of chromium on fungi from forest soils. Fungal communities were examined using PCR-Denaturing Gradient Gel Electrophoresis (DGGE) profiles and fungal-specific primers to monitor changes over time. Chromium bioavailability was measured to determine if any links exist between changes in community structure and metal availability. After Cr was incorporated (0, 200, and 1000 ppm), the bioavailable fraction decreased rapidly and then continued to decline over the 185 day experiment. Fungal community characterization was carried out before, and at 5 and 185 days after, Cr additions. The DGGE profiles of the control soils did not change throughout the experiment. The Cr contaminated soils did exhibit modified DGGE profiles, suggesting that fungal community structure is altered in the presence of metal contamination, similar to bacterial communities. In addition, the DGGE profiles observed before contamination were not found after metal bioavailability had decreased, signifying that permanent changes in the fungal community had occurred after Cr exposure.

 Back to top

32 - Environmental Microbes can Speciate and Cycle Arsenic

E. Danielle Rhine, E. Garcia-Dominguez, C.D. Phelps and L.Y. Young

New York University SBRP

Naturally occurring arsenic minerals in groundwater worldwide has led to increased concerns regarding health risks. Predominantly found in the environment as arsenate [As(V)] or arsenite [As(III)], arsenic can be transformed by microorganisms through oxidative and reductive processes. Oxidation of As(III) to As(V) coupled to O2 and NO3- reduction is energetically favorable and can be used by chemoautotrophic bacteria to fix CO2 for cell growth (Oremland and Stolz, 2003). As(V), however, can be utilized as the electron acceptor in respiration by various anaerobic microorganisms resulting in its reduction to As(III) (Oremland and Stolz, 2003; Liu et al., 2004). Due to the recent description of arsenic-active microorganisms isolated from the environment, we hypothesized that naturally occurring microorganisms could be actively cycling arsenic. We tested this hypothesis using an As(V) respiratory reducer and an aerobic chemoautotrophic As(III) oxidizer. Each strain was grown separately and together in sealed serum bottles, and the oxic/anoxic conditions were periodically changed. Initially, under anaerobic conditions when both isolates were grown together, 2mM As(V) was stoichiometrically reduced to As(III) within 14 days. Following complete reduction, sterile ambient air was added and within 24 hours As(III) was completely oxidized to As(V). The anoxic-oxic cycle was repeated, and sterile controls showed no abiotic transformation within the 28-day incubation period. This work shows that arsenic cycling by microbial activity in response to environmental conditions may affect the speciation, and hence mobility and toxicity of arsenic in the environment.

 Back to top

33 - A simple hydride generation CE-ICP-MS interface for arsenic speciation

Douglas D. Richardson, Rodolfo G. Wuilloud, Sasi S. Kannamkumarath, and Joseph A. Caruso

University of Cincinnati SBRP

Capillary electrophoresis (CE) coupled with inductively coupled plasma mass spectrometry (ICP-MS) has become a popular technique for the separation, identification, and quantitation in elemental speciation. One common drawback from this technique is the poor sample introduction efficiency of the CE-ICP-MS interface that limits the sensitivity by ICP-MS detection(1). Hydride generation (HG) coupled to ICP-MS has been reported to improve detection limits. The formation of volatile hydrides results in a more efficient element transportation to the plasma and better sample ionization. Previous research has shown that CE-HG-ICP-MS is a reliable technique for the speciation of hydride forming elements (2). However, the detection limits were not as low as those obtained with the conventional HG-ICP-MS technique (1). In this work an alternative CE-HG-ICP-MS interface for arsenic speciation is described. Optimization of the variables involved in the HG system was performed to obtain low dispersion of the transient signals generated by the CE device. Analytical figures of merit with the proposed interface were determined and compared with those obtained by CE-ICP-MS. 1. Sasi S. Kannamkumarath, Katarzyna Wrobel, Kazimierz Wrobel, Clayton B'Hymer, Joseph A. Caruso, "Capillary Electrophoresis - Inductively coupled plasma-mass spectrometry: an attractive complementary technique for elemental speciation analysis" Journal of chromatography A, 2002, 975, 245-266. 2. Magnuson M L; Creed J T; Brockhoff C A Speciation of selenium and arsenic compounds by capillary electrophoresis with hydrodynamically modified electroosmotic flow and on-line reduction of selenium(VI) to selenium(IV) with hydride generation inductively coupled plasma mass spectrometric detection. ANALYST (1997 Oct), 122(10), 1057-61.

 Back to top

34 - Analysis of Organophosphorus Chemical Warfare Degradation Products by HPLC-ICP-MS

Douglas D. Richardson, Baki B.M. Sadi, and Joseph A. Caruso

University of Cincinnati SBRP

Recent increases in terrorist activity and the threat of chemical weapon attacks has lead to the demand of a rapid and reliable method for analysis of chemical warfare agents (CWA) and their degradation products. The nerve agents Sarin and VX as well as many others pose a deadly threat to the human population if released. These phosphorus containing nerve agents along with their degradation products present difficulties for ultra-trace analysis due to their low volatility and lack of a good chromophore. Previous studies have successfully utilized methods such as gas chromatography/mass spectrometry (GC-MS), ion mobility/mass spectrometry (IMMS), and liquid chromatography/mass spectrometry (LC-MS) for the analysis of organophosphorus containing degradation products with detection limits in the ng/mL range(1-3). However, these studies did not report detection limits at the more desirable lower levels. Phosphorus analysis by inductively coupled plasma mass spectrometry (ICP-MS) coupled with ion-pairing reversed phase high performance liquid chromatography (IP-RP-HPLC) has recently been proven as a rapid and reliable speciation technique with detection limits in the ng/L range(4). Monitoring 31P with ICP-MS is a difficult task due to its high ionization potential (10.5 eV) as well as the presence of polyatomic interferences, primarily 14N16O1H+ and 15N16O+ which overlap the only phosphorus isotope, 31P. In order to minimize these interferences an octopole reaction cell with He gas is utilized to obtain the maximum signal-to-noise ratio for the samples of interest. In this work a novel method for the rapid analysis of phosphorus containing Sarin (GB) and VX hydrolysis products by IP-RP-HPLC-ICP-MS is described. Analytical figures of merit consisted of detection limits less than 28.0 ng/L with precision values of less than 7% (RSD) for peak area and less than 1% (RSD) for retention times. (1) Steiner, W. E.; Clowers, B. H.; Matz, L. M.; Siems, W. F.; Hill, H. H., Jr. Analytical Chemistry 2002, 74, 4343-4352. (2) Smith, J. R.; Shih, M. L. Journal of Applied Toxicology 2001, 21, S27-S34. (3) Black, R. M.; Read, R. W. Journal of Chromatography, A 1998, 794, 233-244. (4) Sadi, B. B. M., Vonderheide, A.P. , and Caruso, J.A. Journal of Chromatography, A 2004, In Press.

 Back to top

35 - Multisubstrate biodegradation of polycyclic aromatic hydrocarbons in porous media biofilms

Sylian J. Rodriguez, Paul L. Bishop

University of Cincinnati SBRP

Complex mixtures of polycyclic aromatic hydrocarbons and other organic compounds are usually present in contaminated sites. This may influence biodegradation patterns and changes in biofilm growth and development. Biodegradation studies of naphthalene, phenanthrene and pyrene were conducted in sole-substrate systems and in binary and tertiary mixtures to examine substrate interactions on biofilms in porous media systems. It was shown that phenanthrene and pyrene could not be degraded as sole carbon sources in the system, but binary systems of the 3 and 4-ring PAHs with acetate and naphthalene supplements stimulated their degradation, with up to 87.9% and 70.1% removal efficiencies respectively. However, in the tertiary systems the presence of phenanthrene inhibited pyrene degradation. Adsorption of PAHs to sand media was determined to be negligible. Biofilm growth, development and changes in composition were analyzed over time; these showed increases in both firmly and loosely attached viable biomass, as well as extracellular polymeric substance production that formed a complex matrix. Heterogeneous surface films and a variety of biological aggregate structures and growth patterns were observed by confocal microscopy.

 Back to top

36 - Develop mulch biofilm barriers for PAH contaminated groundwater

Y. Seo, Paul Bishop

University of Cincinnati SBRP

PAHs are major recalcitrant compounds in oil contaminants and are known to be carcinogenic to humans. They are strongly bound to soil and are immobile because of their low solubility. However, their dissolution in groundwater is sufficient enough to contaminate large amounts of groundwater over a long period of time (Luthy et al., 1994). Therefore, during remediation of PAH contaminated sites, the slow PAH biodegradation rates may allow contaminant migration through groundwater flow. Because of this, the developments of remediation technologies with new supporting materials which have ability to adsorb pollutants, are low cost and allow design flexibility are needed. Permeable reactive barriers may provide these requirements. In this study, organic mulch was considered as a new supporting material for preventing migration of PAHs and was also used as a medium for biofilm growth. To monitor the fate and transport of the PAH, four lab scale mulch-biofilm column reactors were used to simulate a subsurface permeable reactive barrier under aerobic conditions. Naphthalene was chosen for a model PAH and was supplied with the loading rate of 0.1kg/m2day. A mineral salts solution tank was connected to a saturated naphthalene tank (200 mg/L), and the combined solution was pumped to the bottom of the column at a constant flow rate of 2.55 mL/min., which simulated 5 m/day linear velocity. During the initial operation period up to 400 pore volumes, the removal efficiency had kept stable over 99 % due to sorption and biodegradation. After that, mulch biofilm barriers showed 72~97 % removal efficiency.

 Back to top

37 - Using passive integrative sampling devices to assess the influence of seasonal and episodic events on the distribution of bioavailable PCBs and organochlorine pesticides in the Portland Harbor Superfund site, Oregon

Doolalai Sethajintanin, Robert A. Grove, Kim A. Anderson

Oregon Health and Science University SBRP

Understanding the influence of seasonal and episodic events on the distribution of bioavailable organic contaminants in the river system is necessary in risk assessment/management. However, most conventional water sampling techniques often fail to detect episodic contamination and the real bioavailable fraction of contaminants. The passive time-integrated sampling technique i.e., semipermeable membrane devices (SPMD) is a more relevant approach for prediction of toxicity exposure and risk to aquatic organisms. This in situ membrane-based passive sampler mimics the passive processes of organic contaminant diffusion through biomembrane and partitioning into the organism's lipids. In this study, SPMDs were used to study the influence of seasonal and episodic events on the distribution of bioavailable PCBs and organochlorine pesticides (p,p'-DDT and its derivatives) at Portland Harbor, Oregon (RM 1-18.5), including the Portland Harbor Superfund site (RM 3.5-9.5) and the McCormick and Baxter Superfund site (RM 7). The Harbor sediments have been contaminated from historical or current upland industrial activities. The samplers were deployed within the main river channel near stream outlets of various upstream land uses during different river condition in 2001-2003. Preliminary data indicate that although bioavailable PCB and DDT river concentrations are influenced by historical upland activities, seasonal and episodic precipitation volumes and river flow rates are also important. Bioavailable PCB and DDT concentrations were greater in low river flow/low precipitation conditions than in high river flow/high precipitation conditions. The episodic events (i.e.,unusual high river flow/high precipitation) significantly increased bioavailable PCB and DDT load to the 18-mile stretch of the study area.

 Back to top

38 - Tools for Assessing N-nitrosodimethylamine (NDMA) Biodegradation by Monooxygenase Enzymes

Jonathan O. Sharp, Lisa Alvarez-Cohen

University of California, Berkeley SBRP

The water contaminant N-nitrosodimethylamine (NDMA) is a probable human carcinogen introduced into the environment by the release of rocket fuels and through chlorine-based disinfections of water and wastewater. Previous studies have indicated that bacteria expressing monooxygenase enzymes with relaxed substrate specificity have the potential to biodegrade this compound. We have reported that expression of the soluble methane monooxygenase (sMMO) in Methylosinus trichosporium OB3b, the propane monooxygenase (PMO) in Mycobacterium vaccae JOB-5, and the para toluene monooxygenases (T4MO and TpMO) found respectively in Pseudomonas mendocina KR1 and Burkholderia pickettii PKO1 results in microbial degradation of NDMA. Furthermore, recombinant Escherichia coli containing wild-type derived T4MO and TpMO plasmids can metabolize this compound. In this poster, we describe several simple tools that can be used to screen for relevant monooxygenase activity. The first involves a colorimetric naphthol-diazo assay that has commonly been used to assess the viability of sMMO enzymes. The second tool involves the temporary exposure of the bacteria to a small amount of acetylene gas to serve as an inactivator of monooxygenase activity. Strains OB3b, JOB-5, KR1, and PKO1 were all able to oxidize naphthalene to naphthol as indicated by the colorimetric assay. In each of these cases, this oxidation as well as the oxidation of NDMA was inhibited by the addition of acetylene gas. We report here a positive correlation between the combination of these tools and NDMA biodegradation by a variety of bacterial cultures.

 Back to top

39 - The effect of seasonal and episodic events on biologically available polycyclic aromatic hydrocarbons in the Willamette River, Oregon

Gregory J. Sower, Kim A. Anderson, Robert A. Grove

Oregon Health and Science University SBRP

Concentrations of biologically available EPA priority pollutant polycyclic aromatic hydrocarbons (PAHs) along 18 miles of the lower Willamette River were determined by sampling water with passive sampling devices and analyzed using HPLC with diode array and fluorescence detectors. The study area includes the Portland Harbor (approx. river mile (RM) 3.5 - 9.5) and McCormick and Baxter (RM 7) Superfund sites. Designated as a Superfund site in 2000, Portland Harbor is a heavily industrialized stretch of the Willamette contaminated with heavy metals, polychlorinated biphenyls (PCBs), dioxin/furans, pesticides, and PAHs. Within Portland Harbor lies the former wood treating site of McCormick and Baxter which was designated a Superfund site in 1994 and is characterized by soil, sediment and water contamination by PAHs, penta-chlorophenol, and heavy metals. Preliminary data demonstrate that concentrations of dissolved bioavailable PAHs vary depending on several factors including, but not limited to, precipitation and river flow levels. The effects of increased precipitation and the resulting dilution, runoff, and sediment disturbance must be considered when determining the impact on total contaminant load and concentrations.

 Back to top

40 - Development of a genome-wide screening method to identify gene candidates involved in the degradation of halogenated hydrocarbons using ion chromatography

Sarah Strycharz, Lee Newman

University of Washington SBRP

As the use of phytoremediation for cleanup of halogenated hydrocarbons becomes increasingly widespread, it is imperative to determine the route of metabolism of such chemicals in plants to increase effectiveness and efficiency of degradation. In humans and other organisms, cytochrome P450s are involved in degradation of the common groundwater contaminant trichloroethylene (TCE). TCE and ethylene dibromide (EDB) are both metabolized by the same primary enzyme in humans, cytochrome P450 2E1. Other potential enzymes that may be involved in TCE degradation in plants include additional cytochrome P450s, peroxidases, dehalogenases, laccases, and reductases. In order to screen for multiple enzyme types, we have developed an assay in which bacterial cultures expressing a commercially purchased tobacco leaf (Nicotiana tabacum) cDNA library can be examined for the ability to degrade a halogenated hydrocarbon, EDB. EDB is being used instead of TCE due to the fact that bromide ion (Br-) is not prevalent in culture media, whereas chloride ion (Cl-) can be found at high levels. Degradation of EDB is measured by detection of Br- release using an ion chromatograph. We hope to narrow the number of possible candidate genes by comparing the ability of cDNA library cultures to degrade EDB, with the ability of a control culture expressing a gene known to degrade EDB.

 Back to top

41 - Bimetallic Nanoparticles for Toxic Organic Dechlorination

Yit-Hong Tee, J. Xu, L. Bachas, and D. Bhattacharyya

University of Kentucky SBRP

Reductive degradation of hazardous organics by zero-valent metals is well documented in the literature. Significant enhancement of dechlorination rates can be achieved by using nanosized metals, and by the incorporation of a 2nd dopant metal (such as, Ni, Pd). Our bimetallic nanoparticle synthesis involved two approaches: (1) direct solution phase synthesis by metal salts reduction, (2) in situ reduction of Fe and Ni or Pd ions captured by polyacrylics supported on microfiltration membrane (PVDF) through ion exchange/chelation. TCE (trichloroethylene) degradation with bimetallic Ni/Fe nanoparticles shows that bimetallic system is more efficient than zero-valent metals in terms of higher degradation rate and formation of non-chlorinated by-products. Product formed were predominantly saturated hydrocarbon, ethane. Higher degradation rate is attributed to the high surface area/reactivity of the nanoparticles, and the role of hydrogenation by the dopant metal (Ni or Pd). The overall TCE degradation rate was found to be a strong function of Ni/Fe ratio. In addition to the rapid degradation (by Fe/Ni) of TCE to ethane, we were also able to achieve complete dechlorination (with biphenyl as the main product) of selected chloro-biphenyls (PCBs) using milligram quantities immobilized Fe/Pd nanoparticles in membrane domain (2nd synthesis method). Our research with nanostructured metals (Fe, Fe/Ni, Fe/Pd) is expected to have significant positive impact on pollution remediation through compact and flexible dechlorination technology development with high reaction rates at room temperature, and significant reduction of metals usage. This project is funded by NIEHS-SBRP and EPA-STAR programs.

 Back to top

42 - 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) Induces Organ-specific Differential Gene Expression in Male Japanese Medaka (Oryzias latipes)

David C. Volz, David C. Bencic, David E. Hinton, J. McHugh Law, and Seth W. Kullman

Duke University SBRP

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous environmental contaminant with well-known adverse effects in fish. To link gene expression with altered phenotype (organ pathology), we initially exploited suppression subtractive hybridization (SSH) as an unbiased screening tool to assess qualitative gene expression changes in brain, liver, and testis of adult male Japanese medaka (Oryzias latipes) exposed for 48 h to a single ip-injected dose of TCDD (10 μg-TCDD/kg-body weight). Across these three organs, SSH identified a total of 335 non-redundant genes. Each organ-specific cDNA subtraction library consisted of a unique gene list and corresponding distribution of biological processes. Based on significant homology to known sequences and frequencies within each library, genes hypothesized to be strongly responsive (42 total) within male medaka brain, liver, or testes were semi-quantitatively screened using replicate cDNA nylon membrane arrays. Of these 42 genes screened, CYP1A mRNA was the only transcript significantly higher in TCDD-exposed brain, whereas 12 transcripts (including CYP1A) were significantly higher in TCDD-exposed liver and 34 transcripts were significantly lower in TCDD-exposed testis. In general, the number, type, and direction of mRNA-level responses were associated with histopathological changes observed: the degree of gene expression response and histopathology similarly increased from brain-to-liver-to-testis. Minimal histopathological changes were observed in the brain, glycogen depletion was observed in the liver, and disruption of interstitia and primary spermatocytes were observed in the testis. Based on these data, brain-, liver- and testis-specific mRNA-level targets in male medaka were identified as promising biomarkers of TCDD-induced toxicity for future investigations.

 Back to top

43 - Kinetics and Products of Contaminant Remediation During In Situ Chemical Oxidation of Contaminated Groundwater

Rachel H. Waldemer, James T. Nurmi, Paul G. Tratnyek

Oregon Health and Science University SBRP

Assessing the feasibility of in situ chemical oxidation (ISCO) using Fenton reagent, permanganate, or persulfate requires accurate kinetic data for oxidation of contaminants. In this study, we measured rate constants for many important contaminants for which kinetic data were not available, including the BTEX constituents (benzene, toluene, xylenes), explosives (TNT, RDX, tetryl), oxygenates (MTBE), and 1,4-dioxane. We then developed quantitative structure-activity relationships (QSARs) that can be used to predict oxidation rates for contaminants that have not been studied previously. Also of concern are the products formed from contaminant remediation by ISCO. We are particularly interested in the 'neurotoxin formation potential' of ISCO, because oxidation of some chlorinated solvents (e.g., perchoroethylene, trichloroethylene, and 1,1-dichloroethylene) by ISCO might form chloroacetaldehyde (CAAD) or related neurotoxic intermediates. A method was developed to assay for CAAD and related compounds and the was applied to samples representing a range of ISCO treatments, but only trace quantities of neurotoxic intermediates such as CAAD were detected. The possible health effects of residual manganese - from ISCO with permanganate as oxidant - will also be discussed.

 Back to top

44 - Different effects of estrogen and an organochlorine pesticide chlordecone on murine immunocyte populations

Fei Wang, Stephen M. Roberts and Eric S. Sobel

University of Florida SBRP

Both chlordecone and estrogen accelerate the development of systemic lupus erythematosus (SLE) in female (NZBxNZW)F1 (NZB/WF1) mice. It has been hypothesized that chlordecone influence autoimmunity in NZB/WF1 mice through estrogenic effects, however low doses of chlordecone that shorten the time to onset of SLE produce little estrogenic effects as assessed through uterine hypertrophy measurements. But low doses of chlordecone might produce estrogen-like effects on immunocytes. To test this, ovariectomized NZB/WF1 mice were treated with 17-beta estradiol (0.025mg/kg/day) or chlordecone (0.5 and 2.5mg/kg/day) using sustained-release pellets implanted subcutaneously. Control mice received pellets with matrix only. Flow cytometric analysis after five weeks of treatment found that estrogen treatment altered the distribution of splenic B cell subsets, with a decrease in immature transitional CD21lowCD24high T1 and an increase in CD21highCD24low marginal zone B cells (p<0.01 by ANOVA). Chlordecone treatment had little effect on B cell subsets. Estrogen increased the expression of the germinal center marker CXCR5 (p<0.05) on B cells and the percent of mature splenic CD19-CD138+ plasma cells (p<0.01), a trend seen only weakly with chlordecone treatment. Also, estrogen treatment decreased CD4 and CD8 T-cell populations (p<0.01) and CD11b+CD11c+ dendritic cells (p<0.05), while chlordecone treatment caused a moderate dose-dependent decrease of T cells, but had no effect on dendritic cells. On the other hand, chlordecone, but not estrogen, increased the level of MHC class II expression on splenic B cells (p<0.05). These results indicate that low doses of chlordecone relevant to autoimmunity, may affect immunocyte populations through different pathways from estrogen.

 Back to top

45 - Aerobic Biodegradation of Polynitro Explosives by Isolated Gram-positive Bacteria

Jennifer L. Weidhaas, Daniel P.Y. Chang

University of California, Davis SBRP

Nitroaromatic and polynitro explosives are common environmental contaminants in the U.S. and internationally. These toxic explosives, such as 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), were some of the most widely used high energy explosives in the 20th century. A chemically related compound 2,4,6-trinitrophenol (TNP), or picric acid, is also a historic military explosive and is still utilized today as an industrial chemical. Munitions containing picric acid were assembled at the former Mare Island Naval Shipyard (MINSY) before its closure resulting in significant soil contamination. Two strains of Rhodococcus opacus bacteria were isolated from explosives contaminated MINSY soil based on their ability to degrade TNP as a sole carbon and nitrogen source. These isolates are capable of mineralizing TNP as demonstrated by production of 14CO2 from 14C-TNP [ring(U)]. These gram-positive organisms were also tested to determine if they could utilize other explosives of environmental significance, namely 2,4- and 2,6-dinitrotoluene (2,4-DNT and 2,6-DNT), 1,3,5-trinitrobenzene (TNB), TNT, RDX, 4-nitrophenol (NP), and 2,4-dinitrophenol (2,4-DNP). It was found that these R. opacus isolates could biotransform TNT, RDX, 2,4- and 2,6-DNT, and 4-NP when TNP was also present as a nitrogen source. Additionally it was found that these bacteria could use 2,4-DNP as a sole carbon and nitrogen source and that TNB could be utilized as a sole carbon source. The demonstrated ability of these isolates to degrade multiple explosives of environmental significance is novel and future research will examine their ability to degrade these contaminants in an engineered bioreactor.

 Back to top

46 - Comparative activation of ER alpha and ER alpha/Sp1 in breast cancer cells by xenoestrogens

Fei Wu, Stephen Safe

Texas A&M University

17 β-Estradiol (E2) and diethylstilbestrol (DES) are potent estrogenic compounds in vivo and activate estrogen-responsive genes and constructs containing estrogen responsive element (ERE) in transfection experiments in cell culture. The estrogenic activity of several synthetic 'xenoestrogenic' compounds were evaluated in transient transfection assay in breast cancer cells, transfected with constructs containing three tandem EREs (pERE3) or GC-rich motifs (pSp13) that are activated by ERα/ERE and ERα/Sp1 respectively. Relatively high concentrations (10-50 μM) on nonylphenol, 2,2-bis(p-hydroxyphenol)-1,1,1-trichlozoethane (HPTE), octylphenol, endosulfan and 2',3',4',5'-tetrachlorobiphenol-2-ol induced transactivation in cells transfected with pERE3 whereas maximal induction was observed by 1 or 10nM E2 or DES. 10nM E2 and higher concentrations (up to 75μM) compounds also induced 4- to 5-fold increase in reporter gene activity in MCF-7 cells, whereas only a 50% increase was observed in cells treated with DES. A series of mutant ERα constructs containing selective deletions and mutations were also used in these assay and the results show that transactivation of DES, E2, antiestrogens and xenoestrogens differentially induced transactivation in MCF-7 cells and other breast cancer cell lines transfected with pSp13. Thus xenoestrogens induced activation of ER α/Sp1 and the results suggest 'unique' in vitro estrogenicity that reflects their unique in vivo biology.

 Back to top

47 - Perchlorate and Nitrate Reduction by Native Microbial Communities in Vadose Soils

Kun Yang, Mamie Nozawa-Inoue, Kate M. Scow and Dennis E. Rolston

University of California, Davis SBRP

Perchlorate contamination, resulting from improper disposal of wastes from rocket propellants and highway safety flares, has been found in groundwater and soils throughout the U.S, especially in California. Perchlorate persists in the environment and its health effects include disruption of thyroid hormone production. Currently, biodegradation is a promising technology to clean-up perchlorate contamination. Previous pure culture and bioreactor studies found perchlorate, an electron acceptor, can be reduced to chloride under anaerobic conditions. However, little is known about the potential of native microbial communities to degrade perchlorate in vadose (unsaturated) soils, a substantial source of groundwater and surface water perchlorate contamination. Our objectives were to measure the effects of electron donors (acetate and hydrogen) and other electron acceptors (nitrate) on perchlorate reduction by native microbial communities in uncontaminated and contaminated vadose soils. Anaerobic microcosms were conducted using soils not previously exposed to perchlorate and soils from a contaminated site in California. Perchlorate reduction in uncontaminated soils was enhanced by adding electron donors (acetate and hydrogen) and the lag time was shorter with acetate than hydrogen. In contaminated soils, hydrogen enhanced perchlorate reduction at faster rates than did acetate. Nitrate addition slightly impeded perchlorate reduction in the presence of hydrogen, substantially impeded reduction with no electron donor added, but enhanced reduction with acetate as a donor. DGGE analysis of PCR-amplified bacterial community 16S rDNA showed that addition of perchlorate to uncontaminated soils enriched previously reported perchlorate-reducing isolates, Dechlorospirillum sp. and Dechloromonas sp. with acetate and Azospirillum sp. with hydrogen.

 Back to top

48 - Development of a Green Fluorescent Protein-based Rat Cell Bioassay for Detection of Dioxin and Related Chemicals

Bin Zhao, Michael S. Denison

University of California, Davis SBRP

Halogenated aromatic hydrocarbons, such as the polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls represent a ubiquitous class of highly persistent and toxic environmental chemicals. Exposure to HAHs, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produces a diverse array of species- and tissue-specific toxic/biological effects, the majority of which are mediated by a ligand-dependent transcription factor, the aryl hydrocarbon receptor (AhR). For proper epidemiological, risk assessment and exposure analysis of TCDD and related HAHs, accurate measurements of these chemicals in the species of interest and in various exposure matrices (biological, environmental, food and feed) is required. However, current high-resolution instrumental analysis techniques are very costly, time-consuming and are impractical for large-scale screening studies. While our previously developed recombinant mouse hepatoma cell line (CALUX) responds to TCDD and related HAHs with the induction of firefly luciferase activity, this system has some limitations including high reagent costs and time for analysis. Here we describe a novel stably-transfected rat hepatoma (H4IIe) cell line containing an AhR-responsive enhanced green fluorescent protein (EGFP) reporter. This CAFLUX (Chemically Activated FLuorescent eXpression) cell bioassay responds to TCDD and other HAHs in a time-, dose-, chemical- and AhR-dependent manner with the induction of EGFP (TCDD LOD = 1pM). This novel rat CAFLUX cell bioassay not only is simpler, more rapid and less expensive than our CALUX bioassay and it allows repeated reporter gene activity in the same cells, but it provides us with a high-throughput bioassay system for large scale screening of sample extracts for the detection and relative quantitation of TCDD-like HAHs.

 Back to top

49 - Desorption of PAHs in Treated Soil from a Slurry-phase Bioreactor

Hongbo Zhu, Michael D. Aitken

University of North Carolina SBRP

Contaminated soil obtained from a former manufactured-gas plant site in Charlotte is being treated in a bench-scale slurry-phase bioreactor. To evaluate whether biodegradation of polycyclic aromatic hydrocarbons (PAHs) is limited by bioavailability, desorption of the PAHs in the treated soil is being studied using Tenax® beads as the sink for the PAHs. Treated soil, reagent water, Tenax® beads and the biocide sodium azide (NaN3) were added to each of three centrifuge tubes. Tenax® beads were removed from the tubes at predetermined times and extracted by methanol, and fresh beads were added. The extracts were then analyzed by HPLC. The recovery of the Tenax® beads was 96.6%. Unlike observations made with untreated, field-contaminated soils, there was no sharp distinction between an apparent rapidly-desorbing fraction and a slowly-desorbing fraction for most of the PAHs. Instead, the desorption rates appeared to decline exponentially with time. For most of the PAHs a significant fraction of the total mass remaining after biological treatment was desorbable over a 35-day period, suggesting that this fraction might still be bioavailable.

 Back to top

50 - The little skate, Raja erinacea, as a model for xenobiotic impacts in elasmobranch fish

Joseph St.George, Ian P. Callard

Boston University SBRP

Our laboratory is interested in potential endocrine disruption induced by early developmental exposure to xenobiotics in elasmobranch fish. We are using the little skate, Raja erinacea, as a model, but few studies of sex differentiation in elasmobranchs exist. As in other vertebrates, primordial germ cells (PCS's) are segregated early in ontogeny from primitive endoderm and migrate into the cortex of the developing gonad via the dorsal mesentery. Due to the absence of definitive molecular markers, only morphological endpoints of gonadal differentiation have been used. In this study, we describe initial work in the development of vasa as a marker for germ cell migration and sex determination in the little skate. vasa is a member of the DEAD box protein family, a group of putative RNA helicases with 8 highly conserved regions. vasa is highly expressed in germ cell lines and differentiated gonads, although it's function and regulation is poorly understood. However, recent studies on the seabream, Spaurus aurata, showed that vasa expression is hormonally (Estrogen, GH, GnRH) regulated, and therefore possibly susceptible to xenobiotic, and a marker for endocrine disruption at the earliest stages of sex determination and development. We have cloned a putative vasa homolog from the little skate, Raja erinacea. The 668 amino acid derived protein sequence has a 54% homology to zebrafish vasa protein and a 57% homology to human vasa protein. We are using a 431 base pair fragment of skate vasa as a probe to investigate normal and xenobiotic impacts on germ cell migration and early stages of sex differentiation and gonadal development in the little skate embryo. Exposure to a heavy metal, cadmium, has a significant impact on sex ratio as determined by the presence or absence of claspers, a male sex characteristic. Use of vasa as a marker of normal germ cell migration and gonadal development may provide insight into the normal developmental processes and the effects of heavy metals on gonadal differentiation.

 Back to top

51 - DDT Effects on Zebrafish Egg and Fry Development: a descriptive investigation

Jennifer Vasquez & Priscilla Toral, Ritesh Ramdhani

Mount Sinai School of Medicine SBRP

An introductory descriptive study was conducted to observe the effects of varying concentrations of dichlorodiphenyltrichloroethane (DDT) on the embryonic development of Zebrafish eggs and fry. The Superfund site of concern is the Hudson River and the persistence of organochlorine in the form of polybrominated chlorine (PBC) in it. [Bopp, et al.] A complex mix of organochlorines, including DDT, contaminates the watershed. These contaminants resist degradation and bioaccumulate in fish, posing a health risk to those who eat fish from the Hudson River. DDT's presence in the Hudson makes it a justifiable candidate for extensive research. Seventeen Zebrafish eggs from a stock tank were placed in five 500-ml. beakers. Each of four beakers received concentrations of .01, .5, .05, and .1 micromolars of DDT; the concentrations ranged from mildly to highly toxic. The fifth beaker served as the control and was not contaminated with DDT. Methyl blue was used as a possible enhancer of DDT in tissues. In the control population, no abnormalities were observed in either eggs or fry. In the experimental group, abnormal development of eggs - both in number affected as well as in severity of effect - rose in accordance with the increased concentration of DDT. Fry mortality also increased as the concentration of DDT was increased. Surviving fry showed enlarged egg sacs, as well as curvature along their tails. The surviving fry appeared to be struggling to swim and maintain buoyancy.

 Back to top

52 - The Effect of Arsenic Exposure on a Urinary Oxidative Stress Biomarker in Bangladeshi Women

Carrie Breton, Mahmuder Rahman, Golam Mahiuddin, Andres Houseman, Quazi Quamruzzaman, David Christiani

Harvard School of Public Health SBRP

An introductory descriptive study was conducted to observe the effects of varying concentrations of dichlorodiphenyltrichloroethane (DDT) on the embryonic development of Zebrafish eggs and fry. The Superfund site of concern is the Hudson River and the persistence of organochlorine in the form of polybrominated chlorine (PBC) in it. [Bopp, et al.] A complex mix of organochlorines, including DDT, contaminates the watershed. These contaminants resist degradation and bioaccumulate in fish, posing a health risk to those who eat fish from the Hudson River. DDT's presence in the Hudson makes it a justifiable candidate for extensive research. Seventeen Zebrafish eggs from a stock tank were placed in five 500-ml. beakers. Each of four beakers received concentrations of .01, .5, .05, and .1 micromolars of DDT; the concentrations ranged from mildly to highly toxic. The fifth beaker served as the control and was not contaminated with DDT. Methyl blue was used as a possible enhancer of DDT in tissues. In the control population, no abnormalities were observed in either eggs or fry. In the experimental group, abnormal development of eggs - both in number affected as well as in severity of effect - rose in accordance with the increased concentration of DDT. Fry mortality also increased as the concentration of DDT was increased. Surviving fry showed enlarged egg sacs, as well as curvature along their tails. The surviving fry appeared to be struggling to swim and maintain buoyancy.

 Back to top

53 - Impact of Mitigation Efforts on Changes in Arsenic Exposure in the Health Effects of Arsenic Longitudinal Study (HEALS)

Habib Ahsan, A. van Geen, Y. Chen, F. Parvez, A. Pfaff, V. Slavkovich, J.H. Graziano

Columbia University SBRP

We evaluated the effect of various mitigation efforts on changes in arsenic (As) exposure from drinking water among 11,754 men and women participating in the Health Effects of Arsenic Longitudinal Study (HEALS) in Araihazar, Bangladesh. Mitigation efforts included: 1) well testing, 2) installation of community wells, 3) individual-level health education and 4) well labeling and village level information dissemination. Interventions 1 and 3 were given to all participants but interventions 2 and 4 were done for 80% of participants. The mitigation effectiveness was assessed by evaluating well usage behavior and urinary As among 10,751 participants at baseline (i.e., pre-intervention) and after 2 years (i.e., post-intervention). Overall, 57% whose wells were unsafe (> 50 μg/L) at baseline switched to another well, compared to 17% of those who had safe wells. Irrespective of other interventions, recipients of intervention 4 were more likely to switch (POR = 1.7, 95% 1.5-1.9) than the non-recipients. Average urinary As levels among unsafe well-users dropped from 403.4 to 294.7 μg/g creatinine between baseline and follow-up but remained essentially unchanged among safe well-users (141.8 to 135.0 μg/g creatinine). The largest drop in urinary As (495.0 to 177.0 μg/g creatinine) was among those who switched to a community well. Other predictors of urinary As reduction included distance to the nearest safe well and duration since switching. We conclude that appropriate mitigation strategies tailored to the population can help As exposure reduction/removal and may alleviate future disease burden associated with As exposure from drinking water in Bangladesh.

 Back to top

54 - Comparative Genomics as a Tool to Design DNA Microarrays for Studying the Response of Ralstonia pickettii PKO1 to Suboxic Conditions

F. Daniel Bost, Jerome J. Kukor

Michigan State University SBRP

Our laboratory is studying the regulation of the tbu regulon of Ralstonia pickettii PKO1, a model microorganism capable of degrading aromatic fuel hydrocarbons and chlorinated ethene solvents under suboxic conditions. The tbu regulon encodes the monooxygenase enzymes necessary for hydrocarbon degradation, and previous studies demonstrated enhanced expression of the catechol 2,3-dioxygenase gene (tbuE) with nitrate under suboxic conditions (< 2 mg/L O2). A DNA microarray-based approach is being employed to understand the compensatory mechanisms of gene expression in PKO1 during hypoxia. In the absence of genome sequence data for R. pickettii PKO1, we relied on the availability of genome sequence data for Ralstonia metallidurans CH34, Ralstonia eutropha JMP134, and Ralstonia solanacearum GMI1000 for microarray design. A set of 37 genes was targeted that included genes from six broad functional categories: denitrification, stress response, solvent resistance, oxidative phosphorylation, exopolysaccharide production, and the tbu regulon. PCR products for each candidate target gene were generated using primers designed from DNA alignments of the respective gene sequences from each of the sequenced Ralstonia genomes. Genomic DNA from R. solanacearum GMI1000 was used to assess PCR primer utility and fidelity. The resulting PKO1 amplicons were purified, cloned, and sequenced. Based on PKO1 amplicon sequence analysis, this methodology produced amplicons with sequence similarities as low as 51.5% (narH of R. metallidurans CH34) to the corresponding Ralstonia genome sequences. This study indicates the efficacy of applying known genome sequence data to access genome information in phylogenetically related microorganisms for which the genome sequence is unavailable.

 Back to top

55 - 2,3,7,8-Tetrachlorodibenzo-p-dioxin Induces Suppressor of Cytokine Signaling-2 in Murine B-cells

Darrell R. Boverhof, Elaine Tam, Allison S. Harney, Robert B. Crawford, Norbert E. Kaminski, and Timothy R. Zacharewski

Michigan State University SBRP

The B-cell, a major component of humoral immunity, is a sensitive target for the immunotoxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) possibly by rendering cells less responsive to antigenic or mitogenic stimulation. Potential mechanisms of TCDD action on B-cells were examined in murine B-cell lymphoma cells (CH12.LX) treated with 3 nM TCDD or DMSO vehicle using sequence verified cDNA microarrays. One transcript that was significantly induced by TCDD was Socs2, a suppressor of cytokine signaling. Changes in Socs2 mRNA levels paralleled that of cytochrome P450 1a1 (Cyp1a1) with maximal induction of 3-fold observed at 4 hrs as determined by quantitative real-time PCR. Socs2 induction appears B-cell specific as no induction was observed in TCDD responsive mouse hepatoma cells or human breast cancer cells. TCDD-mediated induction of Socs2 mRNA was dose-dependent and exhibited the characteristic structure activity relationships observed for the aryl hydrocarbon receptor (AhR) ligands PCB-126, ICZ and BNF. Experiments with cycloheximide and AhR deficient B-cells indicated that Socs2 mRNA induction is a primary effect that is AhR dependent. Western analysis confirmed that Socs2 and Cyp1a1 protein levels were also induced in CH12.LX cells. Promoter analysis revealed the presence of 4 dioxin response elements within 1000 bp upstream of the Socs2 transcriptional start site and a reporter gene regulated by the Socs2 promoter was inducible by TCDD. Promoter activity was also dependent on a functional AhR signaling pathway. These results indicate that Socs2 is a primary TCDD inducible gene that may represent a novel mechanism by which TCDD elicits its immunosuppressive effects.

 Back to top

56 - Signal Pathways of Interleukin-8 Induction by a Highly ortho-Substituted PCB in Human Vascular Endothelial Cell

Sung Yong Eum, Yong Woo Lee, Bernhard Hennig, Michal Toborek

University of Kentucky SBRP

Increased angiogenesis is critical to tumor progression and metastasis. We previously reported that specific polychlorinated biphenyls (PCB) congeners such as PCB 104 induced activation of phosphatidylinositol 3-kinase (PI3K)-dependent signal pathway and expression of vascular endothelial growth factor (VEGF), an angiogenesis factor in human microvascular endothelial cells (HMEC-1) leading to increased tumor metastasis. In this study, exposure of HMEC-1 to PCB 104 promoted induction of interleukin-8 (IL-8), the C-X-C cytokine and another potent angiogenic factor. In addition, treatment with PCB 104 stimulated several signal molecules, including Janus family kinases (Jaks), PI3K and MAP kinases. General Jak inhibitor and specific Jak3 inhibitor, WHI-P153, blocked overexpression of IL-8 mRNA and secreted protein, while specific Jak2 inhibitor, AG490, had no effect. In addition, our results indicate that the PI3K pathway was involved in PCB 104-induced IL-8 expression. Specifically, three MAP kianses such as ERK1/2, p-38 and JNK modulated PCB 104-induced IL-8 secretion. Interestingly, the JNK inhibitor, SP600125, did not reduce IL-8 mRNA overexpression, while this inhibitor blocked IL-8 secretion. These results indicate that PCB 104-induced IL-8 expression and secretion might be regulated through coordinated complex signaling pathway including the Jak3, PI3K and MAPK signaling pathways. Based on these data, we suggest that specific PCB congeners, such as PCB 104, can promote angiogenesis for tumor progression and metastasis. This work was supported by NIH/NIEHS, P42 ES 07380.

 Back to top

57 - Activation of Epidermal Growth Factor Receptor Mediates PCB-induced Matrix Metalloproteinase-3 Expression and Transendothelial Migration of Breast Cancer Cells

Sung Yong Eum, Yong Woo Lee, Bernhard Hennig, Michal Toborek

University of Kentucky SBRP

Vascular endothelial cells form a continuous monolayer which functions as a selective barrier to the passage of cancer cells from the blood stream to the underlying tissues during tumor metastasis. Therefore, endothelial barrier dysfunction has a significant influence on the fate of circulating cancer cells. We have hypothesized that polychlorinated biphenyls (PCB) congeners can activate vascular endothelial cells leading to disruption of endothelial barrier function. In this study, we examined the effects of PCB 104 on breast cancer cell migration through the basement membrane of endothelial monolayer. Matrix Metalloproteinases (MMPs) have been postulated to participate in transendothelial migration (TM) of cancer cells or inflammatory cells. Among the studied MMPs, PCB 104 induced MMP-3 at both mRNA and protein levels through activation of Janus family kinase 3(Jak3) and epidermal growth factor receptor (EGFR). Blockage of MMP-3 enzyme activity using specific MMP-3 inhibitor, NNGH, prevented TM of breast cancer cell; however, this inhibitor had no effects on permeability or adhesion of PCB 104-exposed endothelial cell monolayers. Suppression of Jak3 by siRNA or specific inhibitor, WHI-P154, reduced MMP-3 expression and cancer cell migration as well as phosphorylation of EGFR. In addition, specific EGFR kinase inhibitor, PD168393, markedly prevented MMP-3 mRNA expression in endothelial cells and TM of cancer cell. These results indicate that Jak3-mediated EGFR activation is a main early signaling pathway in PCB 104-exposed human endothelial cells leading to accelerated TM of cancer cells through MMP-3 induction. This work was supported by NIH/NIEHS, P42 ES 07380.

 Back to top

58 - PCB 104 Increases Permeability of Human Vascular Endothelial Barrier Through Induction of Vascular Endothelial Growth Factor via MAP Kinase Pathways

Sung Yong Eum, Yong Woo Lee, Bernhard Hennig, Michal Toborek

University of Kentucky SBRP

Polychlorinated biphenyls (PCB) congeners, a group of worldwide, persistent environment contaminants, are known to cause carcinogenesis and tumor promotion and also may affect the development of tumor metastasis. Because vascular endothelial cells create a selective barrier to the passage of cancer cells, we hypothesize that specific PCB congeners may disrupt endothelial integrity and increase the transendothelial migration of tumor cells. To examine these hypotheses, we elucidated the effects of 2,2',4,6,6'-pentachlorobiphenyl (PCB 104), a representative of highly ortho-substituted non-coplanar PCB congeners, on expression of vascular endothelial growth factor (VEGF) which is known to regulate permeability of endothelial monolayers. Exposure of human microvascular endothelial cells (HMEC-1) to PCB 104 induced endothelial hyperpermeability and markedly increased transendothelial migration of the MDA-MB-231 cells. The VEGF receptor (KDR/Flk-1) antagonist, SU1498, reduced PCB 104-induced cancer cell transmigration as well as hyperpermeability. Three independent MAP kinase inhibitors, PD98059 (ERK1/2 inhibitor), SB202190 (p-38 inhibitor) and SP600125 (JNK inhibitor) also inhibited VEGF expression and cancer cell transmigration. Based on these results, we conclude that PCB 104-mediated elevation of VEGF expression and transmigration of the MDA-MB-231 cells were mediated by the MAP kinase signaling. In addition, the MAP kinase pathway was involved in PCB 104-induced activation of AP-1, a transcription factor implicated in the regulation of VEGF gene expression. These results indicate that PCB 104 may contribute to tumor metastasis by inducing VEGF overexpression that stimulates endothelial hyperpermeability and transendothelial migration of cancer cells. This work supported by NIH/NIEHS, P42 ES 07380.

 Back to top

59 - Obtaining High-Molecular-Weight DNA from Contaminated Soils

William Fusco, Ravindra Paidisetti, Carina Jung, Cynthia Brinkman, Kihwan Kim, Andrzej Paszczynski, Frederick Colwell, and Ronald Crawford

University of Idaho

Extraction of high-quality DNA from soil is a limiting factor in most soil metagenome studies. Commercially available kits and previously published methods exhibit serious limitations for objectives such as the extraction of clean DNA of large fragment size (50-300 kb) critical for the preparation of BAC libraries. Thus, our goal has been to develop a universal method that will dependably provide large DNA fragments from soil that are representative of the microbial community and are pure enough to proceed with downstream molecular manipulations. Our primary objective is to identify new genes or pathways in soil microorganisms involved in toxic pollutant degradation. Thus far, we have isolated high-quality DNA from two contaminated soils: a hydrocarbon-contaminated and a carbon tetrachloride-contaminated soil. We accomplished this by standardizing soil pretreatment and DNA extraction parameters. Pretreatments involved stimulation of indigenous microbial populations to make them more susceptible to removal from soil and subsequent lysis. Processing steps included removal of microbial cells from the soil matrix, purification of these cells, enzymatic or chemical lysis and partial restriction enzyme digestion of released DNA in agarose plugs. This was followed by electrophoretic separation of large fragments of restricted DNA, electroelution of clonable DNA from gels, and optimization of cloning parameters for the production of BAC libraries. Here we describe this useful protocol to remove microbial cells from soil and prepare from those cells high-molecular-weight chromosomal DNA of purity sufficient for downstream molecular manipulations such as PCR, restriction enzyme digestion, and cloning (supported by NIH grant 5 R21 ES012814-02).

 Back to top

60 - Dietary Fat Interacts With PCBs to Induce Changes in Lipid Metabolism in LDL Receptor Deficient Mice

Bernhard Hennig, Gudrun Reiterer, Michal Toborek, Sergey V. Matveev, Alan Daugherty, Eric Smart, Larry W. Robertson

University of Kentucky SBRP

There is evidence that dietary fat can modify the cytotoxicity of polychlorinated biphenyls (PCBs), and that coplanar PCBs can induce inflammatory processes critical in the pathology of vascular diseases. To test the hypothesis that the interaction of PCBs with dietary fat are dependent on the type of fat, LDL-R-/- mice were fed diets enriched either with olive oil or corn oil for 4 weeks. Half of the animals from each group were injected with PCB 77. VCAM-1 expression in aortic arches was non-detectable in the olive oil-fed mice, but was highly expressed in the presence of the PCB. PCB-treatment increased liver neutral lipids and decreased serum fatty acid levels only in mice fed the corn oil-enriched diet. PCB treatment increased mRNA expression of genes involved in inflammation and apoptosis in all mice. Dietary fat-dependent differences were detected in the PCB-mediated induction of oxidative stress related genes, suggesting an aggravated oxidative stress in corn oil fed mice receiving PCBs. Feeding both olive and corn oil diets induced genes involved in fatty acid degradation, however, with the upregulation of different key enzymes. Genes involved in fatty acid synthesis were only reduced in corn oil-fed mice, whereas lipid transport/export genes were altered in olive oil-fed mice. These data suggest that dietary fat can modify changes in lipid metabolism induced by PCBs in serum and tissues. These findings have implications for understanding the interactions of nutrients with environmental contaminants on the pathology of inflammatory diseases such as atherosclerosis.

 Back to top

61 - Quantifying Gene Expression to Predict Reductive Dechlorination by Dehalococcoides spp.

Victor F. Holmes, Johnson, D. R., Lee, P. and Lisa Alvarez-Cohen

University of California, Berkeley SBRP

In situ bioremediation is a economically and environmentally desirable remediation alternative for groundwater contaminated with the chlorinated solvents Perchloroethene (PCE) and Trichloroethene (TCE). To date, only bacteria of the genus Dehalococcoides have been reported to degrade PCE or TCE, via a reductive dechlorination pathway, past the carcinogenic intermediate vinyl chloride (VC) to the non-toxic gas ethene. Currently, molecular assessment of the remediation potential of indigenous field site microbial communities is been limited to PCR-based detection of Dehalococcoides spp. 16S rDNA. This method is lacking, however, because the mere presence of Dehalococcoides spp. does not assure contaminant degradation; Strains of Dehalococcoides with identical 16S sequences each carry out only part of the PCE to ethene dechlorination reaction. This project develops molecular tools based on the presence, quantity, and expression of functional reductase genes to assess the degradation activity of indigenous Dehalococcoides spp. and potentially predict the relative rates at each degradation step so that accumulation of toxic intermediates can be avoided. These tools have been applied to characterize a dechlorinating laboratory enrichment culture from the Alameda Naval Air Station (ANAS). Clone library analysis revealed only one Dehalococcoides 16S sequence, most similar to Dehalococcoides strain VS. PCR with primers specific to known reductases revealed genes similar to the tceA and vcrA reductases. Measurement of 16S and reductase copy number using quantitative PCR should provide further insights into the number and ratio of Dehalococcoides strains in this community. Application of these techniques for assessment of field site communities will be discussed.

 Back to top

62 - XRCC3, APE1, XPD, XRCC1 Polymorphisms, Smoking and Arsenic Exposure and Bladder Cancer Risk

Margaret R. Karagas, Angeline S. Andrew, Heather Nelson, Daniel P. Casella, Alan Scned

Dartmouth College SBRP

Exposure to environmental chemicals plays a significant role in initiation of bladder cancer. Partly due to variations in individual susceptibility, however, only a fraction of all individuals exposed to an environmental risk factor will get cancer. Reduced DNA repair capacity can be attributed to mutations or polymorphisms in critical DNA repair enzymes, and can enhance individual susceptibility to endogenous or exogenous sources of DNA damage. To test the hypothesis that polymorphisms in DNA repair genes may influence bladder cancer susceptibility, we analyzed DNA isolated from the blood samples of 355 cases and 559 controls enrolled in a population-based study of bladder cancer in New Hampshire. We found a significantly elevated risk among XPD codon 751 homozygote variants, particularly among never smokers (adjusted odds ratio (OR) = 2.5; 95% confidence interval (CI) = 1.0-6.2) (gene-smoking interaction p<0.04). When we evaluated the independent and joint effects of two XPD polymorphisms, we found a significantly increased risk among individuals with variant alleles for XPD codon 312 (adjusted OR = 5.2; 95%CI = 3.0-9.0) and codon 751 (adjusted OR = 3.6; 95%CI = 2.2-6.3) relative to individuals homozygous wild type for both polymorphisms (gene-gene interaction p<0.001). Individuals with a variant allele in either XRCC3 or APE1 appeared to be at reduced risk compared to WT/WT (XRCC3 241 Thr/Thr, APE1 148 Asn/Asn, adjusted OR = 0.4; 95%CI = 0.2-0.8) (gene-gene interaction p<0.02). Bladder cancer risk was increased among individuals with a variant XRCC3 allele and toenail arsenic levels in the top exposure decile (toenail arsenic level above 0.194 mcg/g, adjusted OR = 2.3; 95%CI = 1.0-5.6) compared with the bottom exposure quartile (gene-arsenic interaction p<0.01). These results support the hypothesis that common polymorphisms in DNA repair genes modify bladder cancer risk and emphasize the importance of understanding the overall genetic and exposure profiles of individuals to assess susceptibility to complex phenotypes such as cancer.

 Back to top

63 - Shifts in Dioxygenase Gene Populations during Polycyclic Aromatic Hydrocarbon Degradation

Sin'ad M. N'Chadhain, Karen Pesce, Jerome J. Kukor, and Gerben J.Zylstra

Michigan State University SBRP

The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. Many pure cultures have been isolated and characterized for their ability to grow on PAHs such as naphthalene, phenanthrene, and pyrene. Using the wealth of genetic information available for PAH degradation, and on the genes encoding the initial PAH dioxygenases in particular, we have designed generic PCR primers targeting that gene fragment encoding the Rieske iron sulfur center common to all dioxygenase enzymes. These Rieske primers were employed to track shifting dioxygenase gene populations in soil following exposure to various PAHs. Creosote contaminated soil was obtained and used to establish enrichment cultures on naphthalene, phenanthrene, and pyrene. The cultures were monitored to identify the rates and extent of PAH degradation (see Norman, Zylstra, and Kukor poster at this meeting). DNA was extracted from the enrichment cultures following PAH degradation. Rieske gene fragments were PCR amplified from DNA extracted from each enrichment culture and an unamended treatment. Sequencing of the cloned dioxygenase gene fragments showed that different suites of genes were present in soil microbe populations under each enrichment culture condition. The naphthalene enrichment was the least diverse with over half the clones sequenced belonging to one sequence type. The phenanthrene and pyrene enrichments each had three equally dominant sequence types. The Rieske PCR products thus represent a convenient and rapid biomarker for monitoring the microbial response to PAH exposure.

 Back to top

64 - Assessing Contamination to Food Chain During Phytoremediation

Lee A. Newman, Sandy Benson and David Coyle

University of Washington SBRP

Phytoremediation is a process where plants absorb pollutants from the surrounding soil or groundwater via their root systems. In the case of organic compounds, plants can take up and degrade the contaminant within the plant tissues. Trichloroethylene (TCE) research has shown that plants will reduce it to CO2, H2O, and other organic metabolites, such as trichloroacetic acid (TCA) and dichloroacetic acid (DCA). Malacosoma disstria (forest tent caterpillars), which naturally feed on poplars, are defoliants and thus might be affected by the TCE transpired through the leaves or any accumulated metabolites. In this experiment, the insects consumed live poplar tissue dosed with TCE. Over the coarse of its life cycle, no noticeable physiological changes occurred. Between the control, low-dose, and high-dose treatments there was no change in the duration of the larval cycle, in the pupation stage, nor percent of adult emergence. Results show that there is no difference in the ecology or biology of the Malacosoma disstria feeding on trees involved in phytoremediation. This greenhouse study had limitations. High mortality was observed in all treatments due to ventilation in the greenhouse and the trees overgrew containers causing premature senescence. Future plans include repeating this experiment and infesting the trees at a younger age to avoid stress. This research will be beneficial to many sectors of our community for it will provide a conclusion to whether or not contaminants, absorbed during phytoremediation, will be available to wildlife consuming the plant tissue.

 Back to top

65 - Studies at a Field Site to Test Phytoremediation of Groundwater Solvents

Lee A. Newman, Milton P. Gordon, Indulius Muiznieks, Mary Loftfield, Robert Kim

University of Washington SBRP

Phytoremediation is the use of plants to clean up environmental toxins. While many forms of phytoremediation rely on accumulation of the contaminants within the plant tissues, phytoremediation of organic solvents can lead to the breakdown of the contaminant within the plant tissue. However, the metabolism of the plants that do this contaminant breakdown can be directly affected by growing conditions for the plant. Thus, while laboratory and greenhouse studies can give an indication as to plant uptake and/or metabolism of the plants, a true understanding the rates and efficiencies of these breakdown processes can only be gained with field grown plants. However, there are other limitations as field work can have different uncontrolled variables, such as contaminant exposure rates. On our test site in western Washington state, we are able to grow plants under field conditions, yet control contaminant exposure and recovery rates. Tests done with trichloroethylene, perchloroethylene and carbon tetrachloride have shown conclusively that plants can take up and remove these compounds at a rate equivalent to water uptake rates. A variety of plants have been shown to have potential for phytoremediation of groundwater contaminants, including poplars, willows and the evergreen tree Leyland cypress.

 Back to top

66 - Shifts in Microbial Community Composition During Polycyclic Aromatic Hydrocarbon Biodegradation

R. Sean Norman, Gerben J. Zylstra, Jerome J. Kukor

Michigan State University SBRP

Polycyclic aromatic hydrocarbons (PAHs) are widespread in nature and are often considered a potential health risk due to their intrinsic toxicity. In many PAH-impacted areas the biodegradative capability of autochthonous microorganisms has proven an effective method of removing or transforming PAHs, thus reducing health risks. However, to further enhance the process of natural attenuation, much research is needed at the genotypic and phenotypic level to better understand the effect of PAHs on the functional diversity of indigenous microbial communities. The aim of this study was to better understand changes in the functional diversity of a microbial community occurring during the biodegradation of select PAHs. PAH degrading cultures were initiated using source material from a creosote-contaminated site amended with 200 mg/l of naphthalene (NP), phenanthrene (PH), or pyrene (PY) and PAH biodegradation and microbial community composition monitored over time. Results demonstrated that degradation of the individual PAHs paralleled cell growth with substantial reduction in concentrations of NP, PH, and PY occurring by 46, 85 and 250 hours, respectively. Molecular monitoring of enrichment cultures before and after PAH degradation by DGGE and 16S rRNA gene libraries suggested that specific phylotypes of bacteria were associated with the degradation of each PAH. Overall, this study combined with characterization of dioxygenase gene diversity (see poster by Sin'ad N' Chadhain et al.) will provide a more complete understanding of the effect of PAHs on the functional diversity of microbial communities and provide a foundation for using molecular-based strategies for predicting the fate of pollutants in nature.

 Back to top

67 - Association between arsenic exposure and urinary 8-Hydroxy-2'-deoxyguanosine(8-OHdG) in a Bangladesh population

Faruque Parvez, Y. Chen, V. Slavkovich, R. Santella, J.H. Graziano, H. Ahsan

Columbia University SBRP

Arsenic is a known carcinogen, but the mechanism by which arsenic causes cancer is not entirely understood. DNA damage by reactive oxidative species (ROS) is suggested to be one mechanism. We present total urinary arsenic and 8-OHdG data from a population exposed to arsenic in drinking water (mean=88 μg/L, range: 0.5 to 584 μg/L) in Araihazar, Bangladesh. Water and urine samples were collected, three times over a period of 2+ years, from members of a cohort participating in a large epidemiological study. The participants were advised to switch drinking wells if high arsenic (>50 μg/L) was found in their well. Study participants were non-smoking women (n=71) between 21 and 52 years of age (mean=34 years). Urine samples were analyzed for total urinary arsenic and 8-OHdG. Overall, we found that urinary 8-OHdG was positively correlated (r=0.25, p<0.04) with urinary arsenic based on the urine sample collected more recently (within 8 months on average). Among subjects (n=39) who continued to drink water containing high arsenic over the past 2-3 years, the correlation was stronger (r=0.52, p<0.001) than among those (n= 32) who switched to a well with lower arsenic (r=0.31, p<0.09). On a small subset of study participants (n=15) on whom urinary arsenic metabolites were analyzed, there was a significant association between urinary 8-OHdG levels and inorganic arsenic. In summary, our pilot study showed that arsenic exposure is associated with oxidative DNA damage in an arsenic exposed population in Bangladesh and this association can be reversed by consuming arsenic free water.

 Back to top

68 - Gene Expression Pattern Alteration in Largemouth Bass by Endocrine Disruptors

Natalia G. Reyero, David Barber, Kevin Kroll, Kevin Johnson, Marisol Sepulveda, Timothy Gross, Nancy Denslow

University of Florida SBRP

Certain environmental pollutants can alter the normal endocrine function of wildlife, leading to impaired reproduction and development. The chosen model for our research is largemouth bass (LMB, Micropterus salmoides), as this species is known to be sensitive to endocrine disruptors and bio-accumulates hydrophobic xenobiotics. LMB were exposed subchronically through the diet to p,p'DDE (7, 37 and 185 ppm) and dieldrin (0.1, 0.6 and 3 ppm) for up to four months. Using real time PCR, we are beginning to elucidate changes in mRNA expression levels for several genes important for reproduction, including three estrogen receptors, vitellogenin, StAR, Cyp1A, Cyp3A and aromatase. For p,p'DDE, in the liver in both males and females, the ERs decrease (16 fold), except for ER alpha, which increases (up to 30 fold). Vitellogenin and Cyp3A increase and Cyp1A generally decreases. In gonad, the ERs decrease in females, while ER beta and gamma increase in males; StAR increases (up to 16 fold in males) and aromatase decreases. For dieldrin, in the liver the ERs decrease, as well as Cyp1A, while vitellogenin and Cyp3A increase (up to 70 fold in females). In gonad, the ERs increase in females and decrease in males, and StAR and aromatase slightly increase in females. These samples have also been used for suppressive subtractive hybridization in order to obtain LMB genes for microarray analysis to model the changes induced in LMB by the studied pollutants.

 Back to top

69 - Challenges of effective risk communication: Examples from the field

Joan R. Rothlein, Diane Rohlman, Dave Stone, Thomas Arcury, Julie Early, Antonio Marin, Sara Quandt, Linda McCauley

Oregon Health and Science University SBRP

Researchers are frequently faced with the challenge of translating health risk findings to the community, or sharing research findings with individual participants in the study. Messages should involve: trust between the communities and the institutes providing the information; culturally appropriate, sensitive and understandable materials; community involvement in information development and dissemination; acknowledgement of scientific uncertainty; comparisons of community data with baseline or benchmark data and discussion of the benefits and limitations of recommended interventions. We provide two examples of how investigators overcome these challenges in releasing fish advisories and sharing results from studies examining pesticide exposure. Fish advisories developed by state governments for consumption of contaminated fish from both sport and subsistence fishing and commercial sources depend on toxicological benchmark values for carcinogenic (cancer slope factors) and non-carcinogenic (RfDs) health effects in calculation of risk-based consumption limits. These advisories require community involvement in outreach of health risk materials that reflect everyday life choices and experiences. In community-based participatory research it is essential that research results be shared with the community and study participants. It can be especially difficult if the clinical significance of the finding is not known-this is often the case with biomarkers of exposure and health effect. Data sharing with participants is improved by providing baseline measurements from the general population such as biomarker levels reported in the National Health and Nutrition Examination Survey (NHANES) and measurements comparing exposed and non-exposed populations. The response to recommended behavioral changes designed to reduce exposure may be limited when comparative or benchmark levels are not available.

 Back to top

70 - Toxicoproteomics of Solvent Neuropathy

P.S. Spencer, M.I. Sabri, D.D. Tshala-Katumbay, S.B. Hashemi, V.S. Palmer, A. Cranson, D. Sproles, *J.G. Pounds, *R.D. Smith, *J.N. Adkins

Oregon Health and Science University SBRP

Sixteen Chinese reports (1996-2004) of occupational neuropathy in Fujian, Guangdong, Helongjiang, Henan, Jiangsu, and Liaoning blame workplace overexposure to n-hexane, the gamma-diketone metabolite (2,5-hexanedione, 2,5-HD) of which binds to epsilon-amino groups of lysine and cross-links proteins. We used proteomic methods to identify neuroprotein targets of 2,5-HD and its more potent aromatic cousin 1,2-diacetylbenzene (1,2-DAB), both of which induce axonal microtubule segregation and neurofilamentous (NF) axonopathy in rodents. Central and peripheral nervous tissue from rats and mice was treated in vitro and in vivo with 1,2-DAB, 2,5-HD or, as negative controls, their respective protein-non-reactive and non-neurotoxic isomers 1,3-DAB and 2,4-HD. Total lysine content correlated with protein susceptibility to gamma-diketones in vitro, those rich in lysine (NF-H, NF-M, kinesin, dynein) showing greater vulnerability than those with low lysine content (NF-L, beta-tubulin). Proteomic analysis of brain cytosol from mice treated one week earlier with 1,2-DAB or 1,3-DAB was performed in triplicate using cLC-FTICR-MS. Numerous protein changes were observed including, in 1,2-DAB-treated mouse brain, a markedly increased abundance of a ubiquitous protein (stathmin) that promotes microtubule disassembly. Since 2,5-HD increases microtubule stability in vitro, and stathmin gene knock-out is associated with adult-onset axonal neuropathy, this widely expressed phosphoprotein may be a key gamma-diketone target. The rapid segregation of axonal microtubules from NFs induced by direct application of 2,5-HD to sciatic nerve in vivo may reflect a failure to transport NFs, which then accumulate proximally in focal swellings. [Supported by NIEHS grants ES 10338 & ES 011384 and the Oregon Workers Benefit Fund].

 Back to top

71 - Arsenic Interactions with Small Thiol Molecules: Model Reactions for Arsenite and Monomethylarsonous Acid Biochemistry

Anne M Spuches, Harriet Kruszna, Anne M. Rich and Dean E. Wilcox

Dartmouth College SBRP

The toxicity of arsenic is believed to originate from the affinity of As3+ species for cysteine (Cys) thiols (S-) in key target proteins, including enzymes that have catalytically essential Cys residues. In spite of these known biological As-thiol interactions, the solution chemistry of As and simple thiol ligands is not well understood. Consequently, we have undertaken a study to quantify the binding reactions of biologically-relevant As species with a set of simple monothiol and dithiol ligands. The arsenic species being investigated are arsenite (As(OH)3) and monomethylarsonous acid (As(CH3)(OH)2), and the thiols in this study include the monothiol glutathione (GSH) and the dithiols dithiothreitol (DTT), dimercaptosuccinic acid (DMSA), and dihydrolipoic acid (DHLA).

To quantify arsenic binding to simple thiols in solution and thereby provide data (i.e. binding constants) and insight for future studies of As-protein interactions, we are using a combination of colorimetric (UV-visible absorption) and calorimetric (heat flow) measurements. The former indicates the formation of As-thiol complexes by the appearance of charge transfer absorption bands in the near-UV, and the data have been analyzed with SPECFIT fitting software. The latter detects the net heat that is released (or absorbed) upon complex formation, thereby allowing us to quantify the thermodynamics (enthalpy and entropy) of As-thiol interactions. In this study we are able to compare As interactions with mono- and dithiols, quantify differences between arsenite and monomethylarsenite binding to thiols, and determine the bonding differences in a series of vicinal dithiols that form 5-, 6- and 7-membered rings with As3+.

 Back to top

72 - Phylogenetic Characterization of an Anaerobic Microbial Enrichment Capable of Mineralizing Vinyl Chloride to Carbon Dioxide

Stuart E. Strand, Christopher B. Walker, H. David Stensel

University of Washington SBRP

Previously we developed an anaerobic enrichment culture capable of utilizing VC as sole carbon and energy source. However, it was unclear which species of bacteria were actively consuming the VC and which were surviving off of secondary metabolic by-products. Stable-isotope probing allows for culture-independent analysis of microbial systems. The cultures were supplied with 13C-VC and samples removed on days 0, 12 and 24 as the VC concentration declined. The initial community DNA was extracted and 16S rRNA genes amplified by polymerase-chain reaction (PCR) using the universal bacterial primers 8F and 1391r. The product was cloned and analyzed via restriction fragment length polymorphism using the enzymes HhaI and MspI. Unique patterns were grouped into operational taxonomic units and sequenced to determine the phylogenetic composition of the community. This analysis suggested that a Bradyrhizobium japonicum-related species was the dominant member of the community. This result was verified by quantitative PCR using primers specific to B. japonicum. DNA was also subjected to CsCl ultracentrifugation and collected in fractions. Fraction density was determined before purification and subsequent analysis with both partial sequencing and terminal-restriction fragment length polymorphism. The most enriched fraction from day 12 displayed both the characteristic T-RFLP peak of the B. japonicum-related species as well as an identical partial sequence match. However, by day 24 most the enrichment community was present in the most enriched fraction. These results suggest that a B. japonicum-related species is a primary consumer of VC and that the metabolism in the enrichment community is tightly linked.

 Back to top

73 - Mass Balance Study of Trichloroethylene Phytoremediation by a Conifer

Stuart E. Strand, Michael P. Dossett, Milton P. Gordon

University of Washington SBRP

A field experiment was conducted to study the phytoremediation of trichloroethylene (TCE) using Leyland Cypress by providing a mass balance on chloride. Field experiments showed that roughly 75% of the added chloride could be accounted for at the end of the season. Only very small amounts of TCE were lost through transpiration and or diffusion through the tree's tissues, while nearly 67% of the chloride added to the system was accounted for as chloride accumulation in the soil. Uptake of TCE was confirmed with trunk cores, which showed a significant gradient of TCE concentration in the trees. This confirms previous work that has shown gradients with height of TCE in trunk cores of trees in the presence of contaminated water. Dichloroethlyene (DCE), a primary metabolite of TCE, was also present in the trunks, however there was no gradient with height above ground. This is the first mass balance study of TCE phytoremediation using a coniferous tree with measurements of diffusive losses including data from the winter season. We conclude that cypress remediation of TCE can result in significant removal and dechlorination.

 Back to top

74 - In Vivo Kinetics of 1,2-Diethylbenzene and 1,2-Diacetylbenzene in F344 Rats

Karla D. Thrall, Allison M. Cartmell, Richard A. Gies, Hong Wu, Jolen J. Soelberg, Joel A. Klein

Oregon Health and Science University SBRP

Diethylbenzene (DEB) is a moderately volatile, colorless liquid, and is found in gasoline, kerosene, and fuel oils. Exposure to DEB has been shown to cause peripheral neuropathy in rats; the ortho isomer of DEB (1,2-DEB) is generally believed to be the isomer responsible. 1,-2-DEB is assumed to be metabolized primarily by direct oxidation of the ethyl side chain to form 1,2-diacetylbenzene (1,2-DAB). An effort is underway to develop a mathematical model to describe the kinetics of 1,2-DEB and 1,2-DAB in vivo to understand the potential health effects of 1,2-DEB in humans. The current project focused on evaluating the time-course kinetics of 1,2-DEB and 1,2-DAB in male F344 rats following IP injection of 1,2-DEB at 30 mg/ml. An analytical method utilizing LC/MS was developed to evaluate the presence of both 1,2-DEB and 1,2-DAB in biological samples. Verification of the 1,2-DAB metabolite was established using GC/MS. Results indicate that the amount of 1,2-DEB in blood peaked at 2 hr post injection and rapidly declined thereafter. In contrast, 1,2-DAB levels in blood were fairly constant up to 24 hr post injection. Urine concentrations of 1,2-DEB were highest at the first sample collection period (12 hr) and dropped dramatically by 24 hr. However, 1,2-DAB urine concentrations peaked at 24 hr and remained elevated at 48 hr. These data will be used in the development of a pharmacokinetic model to describe the kinetics in rats for extrapolation to humans. (Supported by 1-P42-ES-10338 from the National Institute of Environmental Health Sciences, NIH with funds from EPA).

 Back to top

75 - 2,5-Hexanedione-Induced Adduction of Bovine Neurofilament Protein: Mass Spectrometric De Novo Sequencing, Identification of Endogenous and Exogenous Covalently Modified Sites

Sarah Trimpin, Victor L. Hsu, Peter S. Spencer, and Max L. Deinzer

Oregon Health and Science University SBRP

Adduction relevant to 2,5-hexanedione-neuropathy is dynamic, and chemically sensitive secondary alkylation products can be investigated in detail while maintaining physiologically-relevant conditions. The highly sensitive, secondary alkylation products are consistent with a labile isoindolium-adducted salt, chemically similar to aromatic δ-diketones, providing a link in relative reactivities. It is postulated that charge carrying adducts can be stabilized via ionic interactions with phosphate groups in NFP and phosphorylation of NFPs may play a significant role in stabilizing the 2,5-HD-adducted NFP and altering the net charge of the NFP-complex may cause conformational changes and aggregation. These results suggest that 2,5-HD-induced protein cross-linking may proceed via reactions with additional diketone molecules. This observed time-dependence is consistent with the extended 2,5-HD-exposure period necessary for development of 2,5-HD-related diseases in vivo. A detailed understanding of 2,5-HD-adduction products should yield an improved understanding of mechanisms underlying δ-diketone neuropathy. Mass spectrometric de novo sequencing of bovine NFH yielded primary sequence information (308 aa) and allowed investigation of covalently modified sites. The highest homologies were found with human NFH in the N-terminal region and with dog NFH in the KSP-region of the protein. A methionine located in the C-terminal region homologous only to rat NFH (M810) was identified. This result emphasizes the hypervariability of the KSP- and C-terminal regions of NFPs. Several endogenous phosphorylation sites within the KSP-region of NFH were tentatively established and suggest the involvement of possible kinases similar to bovine NFM. Evidence for cross-linking of NFP-subunits in reaction with 2,5-HD was found by mass spectrometry.

 Back to top

76 - Water Arsenic Exposure and Children's Intellectual Function in Six Year-Old Children in Araihazar, Bangladesh: An Interim Analysis

Gail A. Wasserman, X. Liu, F. Parvez, H. Ahsan, P. Factor-Litvak, A. van Geen, V. Slavkovich, N.J. LoIacono, Z. Cheng, J.H. Graziano

Columbia University SBRP

We recently reported an adverse association between drinking water arsenic and cognitive function in 10 year-old children living in Bangladesh (EHP 112:1329, 2004), where approximately 30-40 million people have been chronically exposed. We are now conducting a cross-sectional study of 230 6 year-old children of parents participating in the same ongoing prospective cohort study examining the health effects of As exposure in 12,000 residents of Araihazar, Bangladesh. We report here results of an interim analysis that included 185 children. Water As and Mn concentrations of tube wells at each child's home were obtained by surveying all wells in the region. Children and mothers came to our field clinic, where weight, height, and head circumference were measured. Children provided urine specimens for urinary As and blood samples for blood Pb. Children's intellectual function on tests drawn from the Wechsler Preschool and Primary Scale of Intelligence (WPPSI) was assessed by summing weighted items across domains to create Verbal, Performance and Full-Scale raw scores. After adjustment for sociodemographic covariates, mother's intelligence and an assessment of the childrearing stimulation available in the home, Full-Scale scores (p < 0.04) and to a lesser extent Performance scores were adversely associated with water arsenic; Verbal scores were not. These findings, consistent with our earlier work in 10 year-olds, add urgency to efforts aimed at eliminating arsenic exposure in Bangladesh and elsewhere.

 Back to top

77 - Analysis of Hydroxylated-PCBs /Biphenyls Using Whole-Cell Sensing System

Shifen Xu, Sapna K. Deo, Sylvia Daunert

University of Kentucky SBRP

The metabolites of polychlorinated biphenyls (PCBs) such as hydroxylated PCBs (PCB-OHs) or hydroxylated biphenyls were identified as environmental contaminants. Various studies showed that some of the PCB-OHs are potent inhibitors of human estrogen sulfotransferase. Detection of these chemicals in the environmental and biological samples could provide first-hand information about their levels and lead to a better understanding of their role in toxicity. To that end, we have developed a whole cell sensing system for the detection of PCB-OHs using the strain Pseudomonas azelaica. The bacteria Pseudomonas azelaica HBP1 can use hydroxylated biphenyls as their sole carbon and energy source. These bacteria contain the hbpCAD genes, which are responsible for the degradation of hydroxylated biphenyls. The expression of these genes is regulated by a regulatory protein encoded by the gene hbpR located upstream from the hbpCAD genes. By fusing the gene of the reporter protein luciferase encoded by luxAB to the regulator-promoter cassette, a whole-cell sensing system was developed. In the presence of inducers, such as hydroxylated biphenyls or PCB-Ohs, the regulatory protein HbpR activates the transcription through the promoter resulting in the expression of the reporter gene. This expression can then be monitored by measuring bioluminescence emission after addition of a substrate for luciferase, namely, n-decanal. This whole-cell sensing system was employed in the detection of 2-hydroxybiphenyl and 2-hydroxy-3',4'-dichlorobiphenyl. A detection limit of 5 x 10-7 M was achieved using this system. We envision that the method developed can potentially be employed as a rapid and sensitive way to monitor the presence of low concentrations of PCB-OHs in environmental and biological samples.

 Back to top

78 - Poly-chlorinated biphenyl (PCB) reductive dechlorination using metal nanoparticles within conductive polymeric films

Shifen Xu, Karthik Venkatachalam, Ame C. de Leon, Vasilis G. Gavalas, Dibakar Bhattacharyya, and Leonidas G. Bachas

University of Kentucky SBRP

Metal nanoparticles, due to their high surface area and faster kinetics, have attracted considerable attention in the reductive destruction of toxic organic compounds like PCBs. The encapsulation of nanoparticles within polymeric membranes prevents aggregation, increases their reactivity, and enables their regeneration. A new method is presented where nanoparticles were formed within conductive polymeric films and subsequently applied to the reductive dechlorination of 2,2',4,4'-tetrachloro biphenyl. The polymeric film consists of polycarboxylic acid entrapped within a polypyrrole matrix where the former provides sites for coordination of metal ions, followed by electrochemical reduction of metal ions to the zero-valent state in situ. The effect of various parameters like membrane thickness, metal loading and polycarboxylic acid loading will be discussed.

 Back to top

79 - Method Development for the Low Level Analysis of Maleylacetone, Fumarylacetone, and Succinylacetone in Human Liver by GC-MS/MS

Melissa D. Zolodz, George Henderson, Peter Stacpoole

University of Florida SBRP

Dichloroacetate (DCA) is both a possible environmental hazard as well as an investigational drug. DCA is present in drinking water as a disinfection by-product. DCA inhibits maleylacetoacetate isomerase/glutathione S-transferase zeta (MAAI/GSTz), an enzyme involved in the metabolism of the amino acid tyrosine, which in turn results in the accumulation of tyrosine metabolites. These metabolites, including maleylacetone, may be involved in the hepatotoxicity and neurotoxicity of DCA. Current published methodology to measure maleylacetone has a detection limit of 200 ng/mL (1.2 pmol/mL), however; the levels of these metabolites are likely present in the fmol/uL range in human tissue, such as liver. As a result, the development of a more sensitive method for quantifying these compounds is required. Tyrosine metabolites including maleylacetone, fumarylacetone and succinylacetone are extracted from liver cytosol, derivatized to their pentafluorobenzyl esters, separated and analyzed via GC-MS/MS. This instrumentation allows for a detection limit of approximately 29 amol/uL, while the method detection limit is approximately 95 fmol/uL. Method validation experiments are currently in progress for the analysis of low levels maleylacetone and succinylacetone from human liver.

 Back to top

80 - Stable isotope probing shows flow of pollutant carbon through a soil microbial community

E. L. Madsen, C.M. DeRito, G.Pumphrey

Cornell University

The goal of this study was to use a molecular technique, Stable Isotope Probing (SIP), to identify biodegrading microbial populations by following the flow of carbon from a pollutant (¹³C-phenol) into soil DNA and through to the active populations in a soil microbial community. Our overall approach sought to link field respiration to microbial populations with and without prior exposure to phenol. The field-based assay involved the release of ¹³C-labeled phenol and unlabeled phenol to soil plots and the subsequent analysis of ¹³CO₂ respiration by gas chromatography/mass spectrometry (GC/MS). Key treatments included eleven 20-ml doses(either ¹³C-or unlabeled 10,000 ppm phenol) applied to 5.3 cm² plots at 1-day intervals prior to a final ¹³C-phenol dose. Significant ¹³CO₂ production (>4 times background) was observed from the soil with no prior phenol exposure. ¹³CO₂ release was boosted by a factor of 20 (to 2.15 mmoles) when multiple additions of unlabeled phenol had been previously applied to the soil. As expected, the greatest mass of ¹³CO₂ released was found in plots where all doses of phenol were ¹³C-labeled. Following DNA extraction from the soil plots, cesium chloride density gradient centrifugation was employed to separate ¹³C-labeled soil DNA from the ¹²C-DNA pool. 16S rRNA genes were amplifiable by PCR from the ¹³C-DNA fractions in only the ¹³C treatments (not those that received unlabeled phenol). Sequencing of 16S rRNA clone libraries prepared from the ¹³C-DNA fraction showed distinctive microbial populations in treatments with prior exposure to ¹²C- and ¹³C-labeled phenol. Adapted populations differed from unadapted populations. The patterns also revealed the identities of primary – and secondary – degrading soil populations comprising food chains involved in system carbon flow.

 Back to top