The 2001 Superfund Basic Research Program and Southwest Environmental Health Sciences Center Annual Science Fair was held on April 27th. There were 54 posters on display during the poster session. This was followed by a presentation by guest speaker, F. Peter Guengerich, Ph.D., entitled, "Reactive Intermediates in Toxciology: Past, Present, and Future". Dr. Guengerich is the Director of the Center in Molecular Toxicology at Vanderbilt University School of Medicine, Nashville, Tennessee.
Variation in N-Acetyltransferase 2 Activity and 4-Aminobiphenyl Genotoxicity.
CA McQueen1, B Chau1, RP Erickson1, RB Tjalkens2 and MA Philbert2. 1University of Arizona, Tucson, AZ; 2 University of Michigan, Ann Arbor, MI. N-Acetyltransferases catalyze the acetylation of aromatic amines. There are two genes, NAT1 and NAT2. In humans, polymorphisms in both genes result in phenotypic variation. Low NAT2 activity is associated with increased risk of arylamine-induced urinary bladder cancer and with higher levels of carcinogen-hemoglobin (Hb) adducts. The hypothesis that lower NAT activity increases the genotoxicity of aromatic amines was investigated in mice. C57Bl/6J mice are rapid acetylators with the NAT2*8 allele. A/J mice, slow acetylators, have the NAT2*9 allele and the congenic B6.A strain has theNAT2*9 allele on the C57Bl/6J background. All three strains have the same NAT1 . Adult male mice received a single oral dose of 120 mg 4-aminobiphenyl (4ABP)/kg or corn oil. After 24h, livers were harvested and frozen. Histological sections (4-5μm) were prepared and 4ABP-DNA adducts detected by immunofluoresence. Increased nuclear fluorescence was observed in all three strains. There was no significant variation by strain. Hepatic NAT activity was measured with the murine NAT2 selective substrate p-aminobenzoic acid (PABA) and with 4ABP. Hepatic PABA NAT activity in C57Bl/6J was 7.74±0.13 nmol acetylPABA/min/mg, 3.05±0.58 for A/J and 4.19±0.58 for B6.A. No significant differences were observed in 4ABP NAT activity. Values ranged from 1.1±0.34 to 1.52±024 nmol acetylABP/min/mg. NAT2 genotype correlated with phenotype for PABA acetylation, but there was no phenotype-genotype correlation with 4ABP. The lack of strain variation in 4ABP NAT activity was consistent with the comparable 4ABP-DNA adduct levels seen in the three strains. The association between NAT2 phenotype and 4ABP-Hb adducts in humans was not seen in mice. Substrate selectivity and amino acid sequence support the functional analogy of human NAT2 with murine NAT1 and vice versa. The contribution of murine NAT1 to 4ABP genotoxicity remains to be elucidated. (ES09812, ES10047 (CAM) and ES08846 (MAP)).
Assessing the Role of Ovarian GSH Levels in Ovotoxicity in Rats.
PJ Devine1, IG Sipes2,3, and PB Hoyer1. Department of 1Physiology, and 2Pharmacology and Toxicology, and 3Center for Toxicology, The University of Arizona, Tucson, AZ, USA. The diepoxide metabolite (VCD) of the occupational chemical 4-vinylyclohexene is ovotoxic in rats following repeated exposures. Repeated daily dosing with VCD for 15d destroys the smallest ovarian follicles. Since VCD acutely reduced hepatic levels of the antioxidant, glutathione (GSH), these studies address if reduced GSH levels are involved in the destruction of ovarian follicles. Thus, immature female Fischer 344 rats (n=3-12 per group) were dosed once or daily for 15d with VCD (0.57mmol/kg, ip) or the GSH synthesis inhibitor buthionine sulfoximine (BSO, 2mmol/kg). Animals were killed 2, 6, or 26h following a single dose, and 2 or 26h following 15d of dosing. Liver and ovarian tissues were collected for determination of GSH levels by HPLC. Also, ovaries were collected following repeated dosing and histologically processed for counting follicles. Following a single dose, both VCD (51±5% of control) and BSO (42±9% of control) reduced (p<0.05) hepatic GSH within 2h, but only BSO reduced ovarian GSH (64±5% of control at 6h, p=0.05). Within 26h, liver and ovarian GSH levels had returned to control levels with either treatment. Two h after the 15th dose, BSO (37±5% of control) and VCD (76±4% of control) reduced hepatic GSH levels (p<0.05), but only BSO decreased ovarian GSH (60±3% of control). GSH levels in 15d tissues were similar to controls 26h after the final dose. Oxidized GSH levels were not affected by BSO or VCD at any time point, suggesting these treatments did not induce oxidative stress. Ovarian small follicle numbers were reduced (p<0.05) in 15d VCD-treated rats, whereas BSO did not affect follicle numbers, even though BSO reduced ovarian GSH content. Thus, these results support the conclusion that alterations in GSH levels within small ovarian follicles is not involved in VCD-induced ovotoxicity, though localized effects within target follicles cannot be excluded. (ESO06694, ESO8979)
Ovotoxicity Induced in Rats By 4-Vinylcyclohexene Diepoxide is Associated with Expression and Redistribution of Cellular BCL-2 Family Members.
Xiaoming Hu1, Patty Christian1, I Glenn Sipes2, 3, and Patricia B Hoyer1,3. Department of 1Physiology and 2Pharmacology and Toxicology,3Southwest Environmental Health Sciences Center, University of Arizona, Tucson, AZ Previous studies have shown that ovotoxicity induced in rats by dosing with 4-vinylcyclohexene diepoxide (VCD) is likely via acceleration of the normal rate of atresia (apoptosis). VCD-induced ovotoxicity is specific for small pre-antral follicles and is associated with increased activity of caspase cascades. The present study was designed to investigate the alteration of expression and distribution of Bcl-2 family member proteins induced by the treatment of VCD in rat small ovarian follicles. Female F344 rats were given a single dose of VCD (80 mg/kg, i.p., 1d; a time when ovotoxicity is not initiated), or dosed daily for 15 days (80 mg/kg, i.p., 15d; a time when significant ovotoxicity is underway). Four hours following the final dose, livers and ovaries were collected. Ovarian small (25-100 m m) and large (100-250 m m) pre-antral follicles were isolated, and sub-cellular fractions (cytosolic and mitochondrial) were prepared. Compared with controls, levels of the pro-apoptotic protein, Bad, were greater in both cytosolic and mitochondrial fractions of small pre-antral follicles collected from 15d VCD-treated rats (cytosol, 1.97± 0.16; mitochondria, 2.20 ± 0.24, VCD/Control, p<0.05). After 15 days of daily VCD-dosing, total cellular anti-apoptotic Bcl-xL protein levels were unaffected in small pre-antral follicles, but its distribution in mitochondrial and cytosolic components was altered (mitochondria, 0.635± 0.08; cytosol, 1.39± 0.14, VCD/Control, p<0.05). Likewise, VCD did not affect protein levels of pro-apoptotic Bax in small follicles on d15. However, consistent with a Bax-mediated mechanism of apoptosis, the relative ratio of Bax/Bcl-xL in the mitochondrial fraction of small pre-antral follicles was significantly increased by VCD dosing (1.62 ± 0.21, VCD/control, p<0.05). Immunofluorescence staining intensity evaluated by confocal microscopy visualized cytochrome c protein in the cytosolic compartment in granulosa cells of pre-antral follicles in various stages of development. Relative to controls, within the population of small pre-antral follicles, staining intensity was less (p<0.05) and presumably more diffuse specifically in stage 1 primary follicles from VCD-treated animals (15d). VCD caused none of these effects in large pre-antral follicles or liver (not targeted by VCD). These data provide evidence that apoptosis in rats (15d of dosing) induced in ovarian small pre-antral follicles by VCD is associated with increased expression of Bad protein, redistribution of Bcl-xL protein and cytochrome c from the mitochondria to the cytosolic compartments, and an increase in the Bax/Bcl-xL ratio in the mitochondria. These observations are consistent with the involvement of the Bcl-2 family of genes in VCD-induced acceleration of atresia.
Alpha-Napthoflavone, an Aryl Hydrocarbon Receptor Antagonist, Reverses 4-Vinylcyclohexene Diepoxide-Induced Ovotoxicity in F344 Rats.
K E Thompson1, I G Sipes2,3, and P B Hoyer1,3. Departments of 1Physiology and 2Pharmacology and Toxicology, 3Southwest Environmental Health Sciences Center, University of Arizona, Tucson, AZ, USA. Repeated dosing of rats and mice with 4-vinylcyclohexene diepoxide (VCD) causes loss of ovarian small preantral follicles. VCD increases expression of the aryl hydrocarbon receptor (AhR) in small preantral follicles, but whether this is associated with VCD-induced ovotoxicity is not known. Thus, this study was designed to investigate the effect of the AhR antagonist, alpha-napthoflavone (ANF), on VCD-induced ovotoxicity. F344 rats (d28; n=8-9 rats/treatment) were dosed daily for 15d with either vehicle control (sesame oil; i.p.), VCD (80 mg/kg), ANF (20 or 80 mg/kg), or VCD and ANF. Ovaries were collected, prepared for histological evaluation, and assessed for follicle counts. Treatment with VCD destroyed (p<0.05) a significant number of primordial (60.6% of control) and primary (64.0% of control) follicles. Concurrent treatment with VCD and low dose ANF (20 mg/kg) also resulted in a significant loss of primordial follicles (79.5% of control; p<0.05); however the high dose of ANF (80 mg/kg) prevented VCD-induced primordial follicle loss (93.8% of control). Interestingly, following treatment with the high dose ANF alone, there were greater numbers of primordial follicles as compared to control (40.5% above control, p<0.05). As with primordial follicles, dosing with high dose ANF and VCD protected primary follicles from ovotoxicity (93.3% of control) and high dose ANF treatment alone resulted in increased numbers of primary follicles as compared to control (28.1% above control, p<0.05). Secondary follicle numbers were unaffected by any treatment. These data show that the AhR antagonist, ANF, can prevent primordial and primary follicle loss induced by VCD. Additionally, ANF appears to delay the normal rate of atresia that occurs in primordial and primary follicles during the dosing period. These results suggest that the AhR maybe involved in the physiological control of atresia as well as VCD-induced ovotoxicity.(ES09246)
Effect of 4-Vinylcyclohexene and its Diepoxide Metabolite on Ovarian Expression of Microsomal Epoxide Hydrolase in B6C3F1 Mice.
EA Cannady1, RL Wade3, CA Dyer3, IG Sipes1, and PB Hoyer2. Departments of 1Pharmacology/Toxicology and 2Physiology, The University of Arizona, Tucson, Arizona, USA; 3Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona, USA. Repeated daily dosing with 4-vinylcyclohexene (VCH), an industrial chemical, causes irreversible destruction of ovarian small follicles in mice. Furthermore, the diepoxide metabolite, VCD, is responsible for this follicle loss. In vivo, VCD is detoxified by metabolic enzymes such as microsomal epoxide hydrolase (mEH). The purpose of this experiment was to investigate whether detoxification of VCD could occur within the mouse ovary. Therefore, mEH expression in isolated ovarian fractions from B6C3F1 mice was evaluated (8 animals/group;n=2) and the effects of dosing with VCH or VCD were determined. Female B6C3F1 mice (d 28) were given a single dose or dosed daily (15 d) with VCH (800 mg/kg;i.p.) or VCD (80 mg/kg;i.p.). Ovaries were removed and follicle fractions were isolated (Fraction 1/F1, small pre-antral follicles, 25-100 m m; Fraction 2/F2, large pre-antral follicles, 100-250 m m; Fraction 3/F3, antral follicles, >250 m m). Total RNA was prepared from the follicles and analyzed by RT-PCR using a real time lightcycler. mRNA encoding mEH was expressed in all follicle types. In F1 follicles (those targeted by VCH and VCD) following a single dose, expression of mEH was increased by VCH (322%) and VCD (136%). After 15 d of dosing, expression was increased 530% by VCD, but was largely unaffected by VCH. Following 1 or 15 daily doses with VCH or VCD, expression in non-target fractions (F2,F3) was not as greatly affected as in F1. Based on steady state mRNA, expression of mEH is likely most increased in F1 follicles following a single dose of VCH or 15 doses of VCD. The greater response in F1 follicles suggests that these selective targets of ovotoxicity may be those most directly involved in the detoxification of VCD. Future studies will determine whether this effect is reflected in mEH enzyme activity.(ES09246,ES06694,ES07019)
Effects of Alkylating Agents on SUMO-1 Protein Conjugation in HEK 293 Cells.
Manza, L. L. and Liebler, D. C. Southwest Environmental Health Sciences Center, College of Pharmacy, University of Arizona, Tucson, AZ 85721.The small ubiquitin-related modifier, SUMO-1 is 101 amino acid ubiquitin-like protein that follows a conjugation pathway similar to ubiquitin, but utilizes SUMO specific enzymes. Unlike ubiquitination, which targets proteins for proteasomal degradation, sumoylation exerts a variety of effects, such as the nuclear localization of RanGAP1, the regulation of ubiquitin conjugation of IkB and the transcriptional activity of p53. Additionally, other studies have demonstrated that stress conditions such as heat shock, UV, alkylating agents, topoisomerase inhibitors, and H2O2, increase the levels of SUMO-1 conjugates in cells. We hypothesize that protein damage by alkylation or oxidation triggers sumoylation. To investigate the effects of protein alkylation on SUMO-1 conjugation HEK 293 cells were treated with iodoacetamide, hydroquinone, benzoquinone, 4-hydroxynonenal, hydrogen peroxide, and Texas Redâ C5 bromocetamide at doses of 10, 50 and 100 mM or equal volumes of vehicle (water/DMSO/ACN) at 37°C and 95% air/5% CO2. After 2 hours, the medium was removed and cells were either trypsinized for determination of viability using Trypan Blue exclusion or lysed for protein assay and western blot analysis. Cell viability decreased in a dose dependent mannner for all treatment groups compared to vehicle controls. Western blots indicated that formation of the 90 kDa RanGap1 SUMO-1 conjugate as well as higher molecular weight SUMO-1 conjugates increased with dose while the low molecular weight SUMO-1 monomer appeared to decrease. The results suggest that alkylating agents and oxidants stimulate symoylation of multiple proteins. Identification of chemically modified and sumoylated proteins is in progress. (Supported in part by NIH grants ES06694 and ES10056.)
Identification of Protein Targets and Metabolites of 1,1-Dce in Bile by ESI-MS-MS.
Jones, J.A.1, Kaphalia, L., Moslen, M.T.2 and Liebler, D.C.3 Department of Chemistry1 and Southwest Environmental Health Sciences Center2, College of Pharmacy, University of Arizona, Tucson, AZ, 85721 and Department of Pathology2, Medical Branch, University of Texas, Galveston, TX 77555. The hepatotoxin 1,1-dichloroethylene (DCE) undergoes bioactivation to form 1,1-dichloroethylene epoxide and 2-chloroacetyl chloride. The latter can covalently bind to glutathione to form S-(2-chloroacetyl) glutathione, which alkylates cysteinyl sulfhydryls to form GSCOCH2-S-cys-protein adducts. The epoxide instead forms S-carboxymethylcysteine adducts. Selective damage to the biliary canalicular membrane is observed in animals upon exposure to DCE. We hypothesize that this damage results from alkylation of critical protein targets associated with the canalicular membrane by electrophilic metabolites of 1,1-DCE. To date, however, the identity of the protein targets and the site of modification remain speculative. We are developing a novel, proteomics-based approach to identify protein targets of xeniobiotics that utilizes ESI-MS-MS in combination with the data reduction algorithms SALSA, developed by our research group, and SEQUEST. Model S-carboxymethylated and GSCOCH2-S-cys-peptide adducts were synthesized and their MS-MS fragmentation patterns characterized. Fragment ions, losses from parent ions and ion pairs were identified that corresponded to the modifications. Rats received 50 m g/kg p.o. of 1,1-DCE and bile was collected at 15 min intervals for 3.5 h after exposure. The samples were analyzed by ESI-MS-MS with data dependent scanning and by SALSA, which identifies MS-MS spectra containing specified fragmentation patterns. Use of SALSA in combination with Sequest and de novo sequencing methods has led to the verification of DCE-metabolites, including a hypothesized cyclic degradation product of S-(2-chloroacetyl) glutathione not previously observed in vivo. In addition, a carboxymethylated peptide was tentatively identified as the C-terminal fragment of phospholipase A2. This data demonstrates for the first time the ability to locate small amounts of adducted proteins in complex samples by using MS-MS fragmentation patterns. This work was supported by NIH grants ES06694 and ES 10056.
Identification of a PPAR-Responsive Element in the BCL-2 Gene.
B D Butts, M M Briehl. Department of Pathology, University of Arizona, Tucson, AZ. Apoptosis is a highly regulated cellular process, which, when deregulated, can lead to many adverse pathologies, including cancer. The transcriptional regulation of genes which can promote or inhibit apoptosis is one important mode of apoptotic regulation. One such gene is bcl-2, whose protein product inhibits apoptosis. In comparison to the extensive research on the mode of action of Bcl-2, the transcriptional regulation of bcl-2 is not as comprehensively studied. Peroxisome Proliferator Activated Receptors (PPARs) are nuclear receptors that have been known to regulate genes related to lipid metabolism. Recent work has shown that PPARs may also regulate genes in such fields as carcinogenesis and apoptosis. In this study, we characterize the functionality of a PPAR response element (PPRE) found 3' of the coding region in the human bcl-2 gene. In gel shift experiments, oligos containing the PPRE from bcl-2 showed a decreased mobility when incubated with in-vitro translated PPAR gamma, while a mutated version did not. Reporter assays further demonstrated the functionality of the bcl-2 PPRE. Cells co-transfected with PPAR gamma and a bcl-2 PPRE-containing luciferase plasmid showed a 3.5-fold higher activity when compared to cells co-transfected with the bcl-2 PPRE luciferase plasmid and the empty parent vector of PPAR gamma (pSG). Interestingly, when cells were transfected with pSG, Northern blot analysis of bcl-2 showed the presence of the inactive, 3.5 kb bcl-2 beta message. However, when cells were transfected with PPAR gamma, the 3.5 kb message was absent, while the active, 5.5 kb bcl-2 alpha message was present. Finally, HCA-7 colon cells transfected with PPAR gamma showed decreased amounts of apoptosis induced by the bile acid deoxycholic acid, compared to cells transfected with pSG.
Adaptation to Oxidative Stress affects Apoptotic Signalling in Dexamethasone-Induced Apoptosis.
M M Briehl1, M E Tome1 and E L Jacobson2. Departments of Pathology1 and Pharmacology and Toxicology2. University of Arizona. Tucson, AZ, USA In this study, we investigated whether glucocorticoid-induced apoptosis of the WEHI7.2 mouse thymoma cells is subject to redox regulation. Shortly after treatment with dexamethasone, a synthetic glucocorticoid, WEHI7.2 cells show an increased production of reactive oxygen species. This increase occurs prior to increases in phosphatidylserine externalization, often considered as early event in apoptosis, and well before significant release of cytochrome c from the mitochondria into the cytosol, considered the committed step in this pathway for apoptosis. WEHI7.2 variants with an enhanced antioxidant defense through selection for resistance to hydrogen peroxide or overexpression of catalase or thioredoxin, are resistant to steroid-induced apoptosis and show a delay or lack of cytochrome c release into the cytosol. A comparison of the redox status of the steroid-resistant variants to that of the sensitive cells has shown that the NADP(H) pool is larger and more oxidized in the resistant cells. Glucose 6-phosphate dehydrogenase activity is increased in the variants, including WEHI7.2 cells that overexpress bcl-2, suggesting that the resistant cells both produce and use increased amounts of reducing equivalents. Glutathione S-transferase is also increased in the variants when compared to that in the parental (sensitive) cells. These data suggest that the steroid-resistant variants are primed to respond to oxidative stress and that an early critical event in steroid-induced apoptosis is subject to redox control.
Tyrosine Phosphorylation and Calcium Regulation of MEKK4.
Z E Derbyshire and R R Vaillancourt. Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ, USA. MEKK4 is a recently identified mammalian kinase, though its function in cellular signaling remains undetermined. Analysis of its amino acid sequence suggests it should be a serine/threonine kinase. To characterize the cellular role of MEKK4, identification of co-purifying proteins would potentially provide insight to its function. To achieve this, MEKK4 was expressed using a baculovirus expression system with a hexa-histidine and FLAG monoclonal antibody epitope in order to attach the protein to a solid Sepharose matrix. This recombinant protein was incubated with cellular extract and proteins from a gel were analyzed using proteomic analysis and confirmed by immunoblotting. Using this process, the calcium regulated protein, annexin II, was found to associate with MEKK4, suggesting that MEKK4 could be regulated by calcium as well. Analysis of the sequence of MEKK4 illustrated three possible sites for tyrosine phosphorylation, a unique observation as other characterized MEKK proteins are regulated by serine/threonine phosphorylion. Our studies then were to investigate if and how this was occurring. Immunoblotting with a phosphotyrosine antibody quickly verified the tyrsoine phosphorylation of MEKK4. The next process was to identify the tyrosine kinase responsible for the phosphorylation. As a 60 kDa band was present in the phosphotyrosine immunoblot, it was hypothesized that this band could be src. An immunoblot of a MEKK4 immunoprecipitation with a src antibody was performed and the presence of src was confirmed. Using kinase-inactive MEKK4 as a substrate, a src immunoprecipitation showed tyrosine phosphorylation of MEKK4. Ongoing studies are aimed at determining which MAP kinase isoform interacts downstream with MEKK4. Our long-term goal is to identify and characterize the upstream proteins in the cell that activate a sequential protein kinase cascade for MEKK4 during calcium exposure.
Modulation of p110 PITSLRE Kinase Activity by Tyrosine Hydroxylase.
N A Sachs and R R Vaillancourt. Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ USA. Tyrosine Hydroxylase (TH) is regulated transiently by the reversible phosphorylation of serines 8, 19, 31 and 40, through various intracellular transduction pathways. The physiological significance of phosphorylation of the four serines remains enigmatic, as pan phosphorylation is not obligatory for catecholamine biosynthesis. The purpose of our study was to identify novel interacting proteins in order to characterize the physiological relevance of the heterogeneous phosphorylation of TH. To characterize proteins that interact with TH after phosphorylation on serine 19, we mutated this amino acid to alanine since it was reported that a serine to alanine mutation promotes a stable interaction between proteins. For the identification of proteins that interact with TH, TH(S19A) was co-transformed with a mouse embryo cDNA library into a host yeast strain. Two clones were identified in the yeast two-hybrid system as amino acids 147-262 of mouse-derived p130 PITSLRE. Characterization with the corresponding human cDNA, in the form of a GST fusion protein, encoding amino acids 1-373 of a splice variant of the a isoform of p110 PITSLRE (Af067514) demonstrate an interaction between TH or TH(S19A) and GST-D p110 PITSLRE. Additionally, in vitro transcribed and translated TH interacts with immunoprecipitated FLAG-p110 PITSLRE from transiently transfected COS-7 cells. TH does not serve as a substrate for p110 PITSLRE in an in vitro kinase assay. Moreover, preliminary data suggest that TH mediates an increase in PITSLRE kinase phosphorylation through a cAMP dependent pathway. This is the first report where TH regulates the activity of a protein kinase.
Akt, an Upstream Kinase that Phosphorylates MEKK3.
D G Adams, N A Sachs and R R Vaillancourt. Department of Pharmacology and Toxicology, The University of Arizona College of Pharmacy, Tucson, AZ, USA. Regulation of cell death and survival is an essential component of molecular toxicology that is controlled, in part, by the serine/threonine protein kinase, Akt. A number of environmental toxicants regulate stress-activated protein kinase pathways, however, the proteins that regulate these pathways have not been well characterized. The protein that phosphorylates and regulates the protein kinase MEKK3, a kinase known to function in stress-activated protein kinase pathways, was unknown. In this report, we show that Akt interacts with MEKK3 and phosphorylates two serine residues in the putative amino-terminal regulatory domain of MEKK3. By using liquid chromatography and electrospray ionization tandem mass spectrometry, we have identified phosphorylation of serines 166 and 337 of MEKK3. Phosphorylation of both serines was localized to the consensus Akt phosphorylation site, RXRXXS/T, within MEKK3. Activation of Akt by IGF-1 in PC12 cells resulted in a two-fold increase in MEKK3 phos-phorylation. In addition, recombinant (His)6FLAG·MEKK3, expressed and purified from Sf9 insect cells, interacts with endogenous Akt from Sf9 cells, as well as PC12 cells. Furthermore, precipitated Akt from these cells phosphorylates recombinant (His)6FLAG·MEKK3. Thus, we have identified Akt as an upstream kinase that phosphorylates MEKK3. The interaction between MEKK3 and Akt provides, for the first time, a link between stress-activated protein kinase pathways regulated by MEKK3 with cell death and survival pathways regulated by Akt. This work was supported, in part, by grants from the NIH (AG18041), the Southwest Environmental Health Sciences Center (P30 ES06694), the Flinn Foundation, the Pharmaceutical Research and Manufacturers of America Foundation, and the American Cancer Society (IRG 110T).
Development and Molecular Characterization of HCT-116 Cell Lines Resistant to the Tumor Promoter and Multiple Stress Inducer, Deoxycholate.
CL Crowley-Weber1, CM Payne1,3, M Gleason-Guzman3, GS Watts3, B Futscher3, C Bernstein1, H Garewal2,3,4 and H Bernstein1,3.Departments of Microbiology & Immunology1 and Internal Medicine2, College of Medicine, University of Arizona, Arizona Cancer Center3, Tucson Veterans Affairs Medical Center4, Section of Hematology/Oncology, Tucson, AZ, USA. Evidence from live cell bioassays show that the flat mucosa from patients with colon cancer exhibit resistance to bile salt-induced apoptosis. Three independent derivative cell lines from the colonic epithelial cell line HCT-116 were selected that exhibit resistance to bile salt-induced apoptosis. These were developed as a tissue culture model of apoptosis resistance. Selection was carried out for resistance to induction of apoptosis by sodium deoxycholate (NaDOC), the bile salt found in highest concentrations in human fecal water. Cultures of HCT-116 cells were serially passaged in the presence of increasing concentrations of NaDOC. The resulting apoptosis resistant cells were able to grow at concentrations of NaDOC (0.5 mM) that cause unselected HCT-116 cells to undergo apoptosis in a few hours. These cells were then analyzed for changes in gene expression. Combined data from DNA microarray, 2-D gel electrophoresis/MALDI-mass spectroscopy, and confocal microscopy of immunohistochemically stained preparations indicated underexpression or over-expression of numerous genes at either the protein or mRNA level. Both novel and previously characterized genes that may play a role in apoptosis and early stage carcinogenesis have been identified as upregulated in these cell lines, including Grp78, Bcl-2, NF-k B, maspin, microsomal GST, Rad 23, pirin, cofilin, and NOS2. Under-expressed proteins or mRNAs included HSP90, CD59, elongation factor 2, and ras family member B. The largest functional category in both the under- and over-expressing groups was signal transduction proteins. This indicates that signal transduction pathways provide multiple points for regulatory changes that are targeted when a cell undergoes chronic stress. Analysis of these resistant cell lines has suggested potential mechanisms by which apoptosis resistance may develop in the colonic epithelium of patients at risk for colon cancer.
Role of Mitochondrial Complexes I & II, Reactive Oxygen Species and Arachidonic Acid Metabolism in Deoxycholate-Induced Apoptosis.
CM Payne1,3, D Washo-Stultz1, H Bernstein1,3, C Bernstein1 and H Garewal2,3,4. Departments of Microbiology & Immunology1 and Internal Medicine2, College of Medicine, University of Arizona, Arizona Cancer Center3, Tucson Veterans Affairs Medical Center4, Section of Hematology/Oncology, Tucson, AZ, USA. Bile acids are promoters of colon cancer; however, the mechanism(s) of action of this tumor promoter are largely unknown. Bile acids induce apoptosis in colon epithelial cells and it is probable that the modulation of apoptosis contributes, in part, to colon carcinogenesis. We tested the hypothesis that mitochondria contribute to DOC-induced apoptosis as an upstream event and that a pro-oxidant state of the cell favors survival. We found that pretreatment of HT-29 cells for 2 or 24 hours with 0.01 or 0.1 m M rotenone, an inhibitor of mitochondrial complex I of the electron transport chain, dramatically protected against apoptosis induced by 0.5 mM deoxycholate (DOC). This concentration is representative of the bile salt concentrations found in the gut of individuals on a high fat diet. We also found that inhibition of complex II of the mitochondrial electron transport chain by 0.1 and 1.0 m M thenoyltrifluoroacetone (TTFA) markedly protected cells against DOC-induced apoptosis. We determined that rotenone treatment resulted in a reduction in oxidative/nitrosative stress by using an antibody against nitrotyrosine residues in conjunction with confocal microscopy. We next determined if the scavenging of ROS by the lazaroid antioxidants, U-74389G and U-83836E, could protect against NaDOC-induced apoptosis. Lazaroid treatment resulted in a sensitization of HT-29 cells to DOC-induced apoptosis. Arachidonic acid is normally released from membranes and generates ROS and other products during its metabolism. Inhibitors of arachidonic acid metabolism (e.g. esculetin, sulindac sulfide, NS-398) also sensitized HT-29 cells to DOC-induced apoptosis. We believe that there are two opposing pathways that determine the ultimate fate of the cell. On the survival pathway is the maintenance of a pro-oxidant state with the maintenance of anti-apoptotic proteins, such as NF-k B, NOS2, COX-2 and LOX. On the pro-apoptotic pathway is the damage to mitochondria resulting in the generation of ROS within mitochondria. The exact mechanisms of colonic epithelial cell death by bile acids are currently being explored.
The Growth of Malignant Keratinocytes Depends on Signaling Via the PGE2 Receptor EP1.
EJ Thompson1, A Gupta2, GT Bowden1. 1Arizona Cancer Center, The University of Arizona, Tucson AZ; 2James Cancer Hospital and Research Institute, Ohio State University, Columbus, Ohio. Recent discoveries shed light on the importance of prostaglandin production in development of skin cancer. Work by Fischer's group demonstrates that skin tumor yield caused by UVB can be decreased by up to 89% by blocking COX-2 with the drug Celecoxib (Mol Carcinog.25: 231-40). A similar study by Pentland et al showed that intervention with oral Celecoxib could decrease new tumor formation by 44% in mice that already have at least one tumor (Carcinogenesis.20: 1939-44). These studies demonstrate the importance of COX-2 in the development of squamous cell carcinoma of the skin. Indeed, there is evidence from Kanzaki's group that prostaglandin production can be essential for the growth of human skin tumor cells in culture (Int. J. Cancer 86: 667-71). We have explored growth signaling in a model of skin tumor progression. Since changes in prostaglandin production have been implicated in skin carcinogenesis we examined this pathway. We have treated the benign murine papilloma producing keratinocyte cell line 308 with ionizing radiation to model tumor progression in vitro. This produced the progressed malignant variant cell line, 6R90. Another cell line was established from an unusually aggressive tumor formed by subcutaneous re-injection of 6R90 cells (6RI). Using enzyme-immunoassay, we found that the malignant variants secrete more PGE2 than the parental 308 cells. We observed alterations in the expression of COX-1 and COX-2 by western blot analysis. We also found that these cells express the PGE2 receptors, EP1 and EP4. When the cells are grown in the presence of indomethacin, the growth rate of the malignant variants, but not the 308 cells, is decreased. This effect can be reversed by addition of PGE2 to the medium. This growth inhibition can also be rescued by addition of an EP1 agonist to the medium. Thus, we have shown that skin tumor cells depend in part on PGE2 signaling via the EP1 receptor for their in vitro growth.
Microarray Technology to Profile CpG Island Methylation in Cancer.
B W Futscher, N Holtan, and R B Isett. Arizona Cancer Center, University of Arizona, Tucson, AZ, USA. The objective of this research project is to adapt microarray-based technology to the measurement of CpG island methylation in human cancer cells. CpG islands are ~1kb stretches of DNA that have a high CG content, are enriched in the dinucleotide 5'-CG -3', are found at the 5' end of ~50% of all human genes, and participate in the transcriptional regulation of these genes. The cytosines in the CpG dinucleotides of CpG islands are unmethylated in normal tissue; however, CpG islands become aberrantly methylated during oncogenesis, and this aberrant methylation has been linked to the transcriptional repression of the associated gene. The target genes of CpG island methylation are often associated with carcinogenesis and include tumor suppressors, metastasis suppressors, and DNA repair genes. In addition, from the limited number of CpG islands and tumors that have been analyzed to date, it appears that patterns of aberrant methylation occur in a tumor-specific and stage-specific fashion, suggesting that CpG island methylation profiles may be useful as a tumor-specific fingerprint to monitor disease activity and burden. Thus, a multiplexed assay where the cytosine methylation status of thousands of CpG islands can be determined simultaneously would be useful in the molecular profiling of human tumors, and will likely provide insights into the biology of cancer. To this end we initiated production of human CpG island microarrays (CGI arrays) as a tool for determining CpG island methylation profiles in cancer, and from these profiles identify characteristic patterns of CpG island methylation that correlate with the tumor's clinical phenotype.
The Ability of P53 to Reverse the Epigenetic Silencing of Maspin in Human Breast Cancers.
RJ Wozniak, MM Oshiro, F Domann, and BW Futscher. Department of Toxicology and Pharmacology, University of Arizona, Tucson, AZ, USA. Arizona Cancer Center, University of Arizona, Tucson, AZ, USA. Maspin is an anti-metastatic, tumor suppressor gene whose expression is inappropriately silenced in breast cancer. The transcriptional silencing of maspin is not due to gene deletion or mutation, indicating that other regulatory mechanisms are likely at work. We have found that aberrant cytosine methylation-associated chromatin condensation of the maspin promoter region is associated with its transcriptional inactivation in human breast cancer cell lines. In addition, recent studies in prostate cancer suggest that wild type p53 is a positive regulator of maspin expression. Based on these observations we set out to test the hypothesis that enforced over-expression of wild type p53 can overcome the repressive effects of aberrant promoter methylation by stimulating chromatin remodeling of the maspin promoter. Three aberrantly methylated, maspin negative human breast cancer cell lines were used to test this hypothesis; two were mutant for p53 (MDA-MB-231 and MDA-MB-435), while one contained wild type expression of p53 (MCF-7). These three cell lines were used as targets for adenoviral infection of a vector containing wild type p53 and green fluorescent protein. Results from the enforced over-expression of wild type p53 resulted in maspin re-expression in all three cell lines, compared to non-infected controls. These results suggest that wild-type p53 can overcome the transcriptionally repressive effects of aberrant cytosine methylation, and suggests that p53 may participate in chromatin remodeling. Future experiments are designed to delineate the potential mechanisms by which p53 elicits these effects.
Microarray Expression Profiles in Breast Cancer Cells Following Exposure to Benzo[a]pyrene and B[a]P-Diol Epoxide.
D F Romagnolo, B D Jeffy, DJ Samuelson, R B Chirnomas, and C M Payne. Department of Nutritional Sciences, Cancer Biology IDP, Microbiology and Immunology, The University of Arizona, Tucson, AZ. Maintenance of genome integrity in mammalian cells may be compromised by the concomitant accumulation of DNA damage and loss of repair functions. Polycyclic aromatic hydrocarbons (PAHs) are classic DNA-damaging and tumor promoting agents. Benzo[a]pyrene, a prototype PAH, induces a number of genotoxic responses including cell cycle arrest, oxidative stress and DNA adduct formation. The objective of this study was use DNA microarray as a means to assess the genome-wide expression profile after exposure to benzo[a]pyrene (B[a]P) and its metabolite 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE). Exposure of MCF-7 cells to benzo[a]pyrene (B[a]P) (1 to 5 mM) and BPDE (50 to 100nM) induced transient S-phase arrest, which was followed by accumulation in G2/M . In cells treated with B[a]P and BPDE, we observed segregation of nucleolar material and a drastic reduction in the number of ribosomal fibrillar centers. To identify the expression profile in cells treated with B[a]P or BPDE, we analyzed the expression levels of 1142 genes by GeneMAPTM CancerArray. Compared with the expression levels in untreated cells, transcripts that were upregulated by both B[a]P and BPDE included members of the cytochrome P450, AP-1 (Fra-1, Fra-2), and glutathione-S-transferase families, PARP, and G1/S specific cyclins. In contrast, transcript levels for BRCA-1, G2/M cyclins, Bcl-2, JunB, and JunD were downregulated. The treatment with the metabolite BPDE tended to elicit a stronger response than B[a]P did. RT-PCR analysis for a subset of genes confirmed the changes in expression levels predicted by DNA microarray.
Regulation of the BRCA-1 Promoter in Breast Cancer MCF-7 Cells by Benzo[a]pyrene and its Metabolite BPDE.
B D Jeffy, D F Romagnolo. Cancer Biology Graduate Interdisciplinary Program, Department of Nutritional Sciences, University of Arizona, Tucson, AZ, USA. The objective of this study was to investigate potential mechanisms underlying the negative regulation of BRCA-1 expression by the PAH benzo[a]pyrene (B[a]P) and its metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE). In previous studies, we found that treatment of MCF-7 breast cancer cells with either B[a]P or BPDE significantly decreased BRCA-1 message and protein levels in a time- and dose-dependent manner. In this study, in order to determine if loss of BRCA-1 message induced by B[a]P or BPDE might be occurring via a regulatory mechanism, we cloned a 1690 bp fragment of the 5¢ BRCA-1 flanking region into a luciferase reporter vector for transient transfection. We found that transfection of the construct (pGL3-BRCA-1) into MCF-7 cells followed by treatment with either B[a]P or BPDE caused a dose-dependent decrease in luciferase activity. However, another ligand of the AhR, TCDD, did not alter pGL3-BRCA-1 luciferase activity. We previously reported that B[a]P exerts its negative effects on BRCA-1 message and protein levels in estrogen receptor positive (ER+) cells but not in estrogen receptor negative (ER-) cells. In order to further characterize the relationship between ER status and regulation of BRCA-1 expression, we transfected pGL3-BRCA-1 into ER- HBL-100 mammary epithelial and ER- HeLa cervical cancer cells. Treatment of these transfected cells with B[a]P caused no decrease in luciferase activity. However, co-transfection of pGL3-BRCA-1 and a plasmid containing estrogen receptor cDNA (pHEO) followed by treatment with B[a]P did indeed cause a reduction in reporter activity. These data suggest that downregulation of BRCA-1 by B[a]P may be occurring via a regulatory mechanism dependent on ER status.
Effects of Occupational Levels of Toluene Diisocyanate on Glutathione-Dependent Enzymes and Glutathione in Human Bronchoepithelial Cells.
R C Lantz1, R Lemus2, D H Wilson2, and M H Karol2. 1Dept. of Cell Biology and Anatomy, Health Sciences Center, University of Arizona, Tucson, AZ and 2Dept. of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA. Toluene diisocyanate (TDI) is a recognized asthmogen, yet the mechanism of its toxicity and the molecular reactions involved remain unclear. We have previously shown a rapid reaction of TDI with glutathione (GSH), a key cellular antioxidant. The intracellular GSH level is the final outcome of several processes, one being the uptake of constituents of the GSH molecule via the glutamyl transpeptidase (g-GT) pathway. This enzyme plays a key role in the synthesis and degradation of GSH. Hence, the activity of g-GT may provide a sensitive indicator of lung injury. Our earlier in vitro studies using the fluorescent dye, Cell Tracker Green (5-chloromethyl-fluorescein diacetate, CMFDA), have shown a decrease in intensity of the thiol staining as a function of the length of TDI exposure in differentiated and undifferentiated human bronchial epithelium (HBE) cells. Because CMFDA activation and fluorescence requires functional intracellular esterases and glutathione-S-transferases (GST), the present study was undertaken to evaluate the activity of these two enzymes as well g-GT activity. Differentiated (6-day) air/liquid cultures of HBE cells were exposed to 20 ppb TDI vapor for 5 or 30 minutes. Following exposure, cell viability was analyzed, cellular GSH levels were titrated using the thiol specific fluorophore ThioGlo, and GST and g-GT enzymatic activities were spectrophotometrically determined. Cellular esterase activity was additionally assayed by placing cells in PBS containing 0.5 mM 5-(and-6-)-carboxyfluorescein succinimidyl ester (CFSE) followed by imaging using confocal microscopy. Exposure of 6-day cultures to 20 ppb TDI for 5 and 30 minutes was not cytotoxic. In addition, esterase and GST activities were not affected by TDI exposure. Therefore, the reduction in CMFD fluorescence detected following TDI exposure was not due to cytotoxicity, decreased esterase activity, nor reduced GST activity. TDI exposure of HBE cells resulted in a 30 % decrease in GSH levels and 35% decrease in g-GT activity. This reduction of intracellular GSH and inhibition of a GSH-dependent enzyme can alter cellular redox status. The resulting oxidative stress may be a significant factor in determining human susceptibility to TDI-induced lung diseases. Supported by NIEHS Center Grant #ES06694 and NIEHS #05651.
Glucuronidation of Bisphenol A in Hepatic Microsomes: Age-Dependent Differences.
Kuester, R.K., Pritchett , J.J., Fontaine, S.M., Solyom, A and Sipes, I.G. Department of Pharmacology and Toxicology, The University of Arizona, Tucson, Arizona Bisphenol A (BPA) is a phenolic compound with industrial and commercial uses. In the liver Bisphenol A undergoes extensive metabolism and is eliminated primarily as a glucuronide conjugate. To understand how age affects BPA glucuronidation, kinetic constants for BPA-glucuronidation (Vmax and Km) were determined in incubations of hepatic microsomes isolated from adult male (77-day old), newborn (4 and 21 day) and fetal (gestational day 19 [GD-19]) Sprague Dawley rats. Microsomes were incubated with UDPGA and 14C-BPA (2.6-402 µM) for 2 min. Reaction products were separated via HPLC and analyzed by scintillation counting. Under the conditions of these experiments the only metabolite obtained was the monoglucuronide of BPA. Fetal hepatic microsomes showed a decreased capacity to glucuronidate BPA as compared to hepatic microsomes of adult rats (38 versus 72 nmol/min/mg, respectively). Glucuronidation of BPA was increased in microsomes from livers of 4-day old rats as compared to those of GD-19 rats. Based on Vmax values, these activities were still lower than those of hepatic microsomes from adult and 21 day old rats. There were no major differences in Vmax values between microsomes from 21 and 77-day old rats. For all age groups the Km values ranged 22 µM to 42 µM. These data demonstrate that the glucuronsyltransferase activity responsible for BPA metabolism is developed before GD-19 and increases soon after birth. Although the activity in GD-19 microsomes was lower than that of adult microsomes, livers from GD-19 represented 9% of body weight as compared to 4% for all other groups. An important consideration is whether this increased liver mass promotes more extensive in vivo conjugation of BPA by fetal hepatic tissue than is predicted from in vitro studies. This research was supported in part by the Southwest Environmental Health Science Center (ES 06694) and the Society of Plastics Industry Inc.
Hepatoprotection during Ischemia/Reperfusion in the Fischer 344 Rat: Comparison of Dimethyl Sulfoxide and Insulin.
CR Kundavaram, MA Leslie, NJ Young, DC Caretto, PZ Nakazato and JB Ulreich. University of Arizona, Department Surgery, Tucson, AZ. UNOS lists 16,000 patients nationwide waiting for liver transplants. Many (12%) will die waiting. Use of non-heart-beating donor organs or enhancement of viability of livers from heart-beating donors might reduce the shortage. Warm ischemia causes damage that can lead to graft failure in recipients. We reported that ischemia/reperfusion in rat livers caused increased adhering leukocytes, swollen endothelial cells and phagocytic Kupffer cells. DMSO pretreatment prevented these effects. Insulin pretreatment has been reported to have protective effects on rat livers during portal triad clamping and improved liver function post-transplantation (Morimoto et al.). To compare the efficacy of DMSO with that of insulin, F344 rats were given DMSO (2 ml/kg, ip), insulin/20% glucose (15ml/kg, iv) or saline (control) 30 min. prior to ischemia. Ischemia (60 min.) was followed by reperfusion (60 min.). Livers were precision-cut and incubated for 4-48h. Following incubation, assays for K+ retention and LDH release indicated viability. DMSO pretreatment maintained K+ levels significantly better than insulin. LDH release by DMSO pretreated livers was lower than for insulin pretreated livers. DMSO prevented loss of viability to a greater extent than insulin. Given prior to organ retrieval, DMSO may preserve organ viability, thus making more organs available for transplantation. In cold preservation solutions, DMSO does not have a similar beneficial effect. In a pilot study using the SELDI (Surface Enhanced Laser Desorption/Ionization) ProteinChip System which shows differential expression of proteins, a protein at 8.31 kDa was upregulated in the presence of DMSO, only under ischemic conditions. The TagIdent database was used to identify potential candidates for this marker. Apolipoprotein C-II (pI: 4.71, MW: 8329.12) was the closest matching candidate. [Supported by ADCRC (JU, PN), UBRP, NIEHS P30-ES-06694 (JU)]
Herbal Remedies Protect against Chloroform Hepatotoxicity in Precision-Cut Tissue Slices.
JB Ulreich, MA Levy, DC Caretto, NJ Young, CR Kundavaram, and PZ Nakazato. Dept. Surgery & Southwest Environmental Health Sciences Center, University of Arizona, Tucson, AZ. By 1990 an estimated 60 million Americans used at least one form of alternative medicine at an estimated cost of $13.7 billion . Trade groups estimate that sales of herbal medicines exceeded $4 billion in 1999. A number of herbs are touted to have beneficial effects on the liver but scientific evidence of their safety, efficacy and mechanism of action is limited or lacking. Precision-cut male Fischer F344 rat liver slices were used to determine whether herbs exhibited protective effects against chloroform toxicity in vitro. Three such herbs were included in this study: Schizandra (Schizandra chinesis), milk thistle (Silybum marianum) and Reishi mushroom (Ganoderma lucidum). Tinctures were concentrated 4x under nitrogen to remove the alcohol and added to culture medium at 0, 1, 5, or 10µL/ml. Livers were excised, cored (8mm), precision-cut (250µ), and incubated at 37oC on titanium carriers in a rolling incubation system for volatiles for 1h in Waymouth's medium +/- each herb. After preincubation, chloroform (80µL/L was injected into some bottles and incubation continued for 2-9h. Potassium and lactate dehydrogenase content of the tissue slices were determined as viability indices. All three herbs showed hepatoprotective effects. Viability (potassium content) of liver slices exposed to herbs with chloroform, compared to chloroform alone, was 129-192% for Schizandra, 129-187% for milk thistle and 143-184% for Reishi, depending on dose. Schizandra and milk thistle incubated together had additive effects. Chaparral (Larrea tridentata), previously reported by us to be a hepatotoxic herb, had potassium values 61-90% of chloroform alone. In vivo studies will be conducted to determine whether there is good correlation with this in vitro model of hepatoprotection. [Supported by NIEHS Center Grant P30ES06694 (JU), and the UA Undergraduate Biology Research Program (CK, DC, NY)].
Determining the Effect of Heavy Metals on Protein Function by Direct Force Measurement. Phase I: Tethering Proteins to Molecularly Smooth Mica in order to Study Protein-Protein Interactions.
S Kim, C Heo, and J E Curry. Department of Soil, Water and Environmental Science, University of Arizona, Tucson, AZ, USA. The overall goal of our work is to directly measure the effect of environmental contaminants such as heavy metals on protein function. More specifically, we want to determine the effect of cadmium on cadherin mediated cell-cell junctions by directly measuring the force between cadherin fragments tethered to solid supports (molecularly smooth mica). In order to create sites for protein attachment we first coated the mica surface with an organosilane monolayer of octadecyltriethoxysilane (OTE). The focus of this study has been to determine if the OTE monolayer is covalently attached to the molecularly smooth mica surface. OTE monolayers were self-assembled on bare and water vapor-plasma treated mica surfaces. The stability of the OTE monolayers on treated and untreated mica surfaces was studied as a function of relative humidity using a surface forces apparatus. Measurements of the thickness of the water layers adsorbed by the OTE were made as a function of relative humidity. The monolayers self-assembled on plasma treated mica adsorbed significantly less water compared to the untreated case. It is thought that the increased stability of the organic layer self-assembled on plasma treated mica is due in part to covalent bonding between the silanes and hydroxyl groups on the mica surface introduced by plasma treatment. This highly stable organic layer will be useful as a base substrate for our cell adhesion studies.
The Effects of Hepatoprotectants, DMSO, Aminobenzotriazole And Cyclooxygenase And Lipooxygenase Inhibitors on Eicosanoid Levels during Chloroform Induced Hepatic Injury.
C.K. Begay, A.J. Gandolfi. Department of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona. Cytochrome P-450 inhibitors Dimethyl Sulfoxide (DMSO) and aminobenzotriazole (ABT) were administered 24 hours after chloroform dosing to determine their effect on eicosanoids levels in rat plasma and liver. Our previous studies have demonstrated that the antidote, DMSO protects against liver injury elicited by chloroform even when given 24 hr after the toxicant, at a time when the liver injury is taking place and rapidly developing. Arachidonic acid is a fatty acid that serves as a substrate for the enzymes leading to the production of potent substances such as prostaglandins and leukotrienes. Their formation in the liver contributes to the acute response to inflammation. DMSO is an anti-inflammatory agent and may affect the production of prostaglandin and leukotrienes. Cytochrome P-450 is involved in biotransforming arachidonic acid to its active metabolites and is inhibited by DMSO. We are now examining if this is one of the mechanisms of DMSO hepatoprotection. Inhibitors for arachidonic acid metabolites were studied to determine what route DMSO or ABT may take in attenuating liver injury. PGE2 and LTB4 are metabolized by the enzymes cyclooxygenase and lipoxygenase, respectively. Both DMSO and ABT prevented an increase in alanine aminotransferase (ALT) at 32 and 48 hr after initial toxicant insult. Plasma PGE2 and LTB4 levels were attenuated by a single dose of DMSO (2ml/kg) or ABT (30mg/kg) to Sprague Dawley rats at 48 hr. Indomethacin, a COX-1 inhibitor, and NS-398, a COX-2 inhibitor both prevented an increase in ALT levels at 48 hr after toxicant ingestion. NDGA, a lipoxygenase inhibitor, Indomethacin, and NS-398 significantly reduced plasma PGE2 levels 48 hr after chloroform dose. Liver PGE2 was not effected by DMSO, ABT or any of the eicosanoid inhibitors. These results show that DMSO and ABT prevented plasma and liver LTB4 formation and provide further support for a role for DMSO as a therapeutic agent against hepatotoxicants. The lipoxygenase pathway may be a major factor in the attenuation of liver injury.
SWEHSC Synthetic Core Laboratory.
Y Li, B Jagadish, and E A Mash. Department of Chemistry, The University of Arizona, Tucson, AZ, USA. The synthetic core laboratory of the South West Environmental Health Sciences Center consults with SWEHSC members on the chemical aspects of their projects and carries out syntheses of labeled and unlabeled compounds. Recent syntheses in support of SWEHSC projects will be presented.
IMPACTT: Integrating Multiple Perspectives for Today and Tomorrow - A Completely Integrated Environmental Health High School Program.
S D Hines, R B R Milholland. Southwest Environmental Health Sciences Center, University of Arizona, Tucson, AZ, USA; Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ, USA. IMPACTT is being developed in partnership with the Sunnyside Unified School District (SUSD) and is a unique, fully integrated environmental health/environmental science academy, or "school within a school." In this first year of the program, fifty 9th grade students are obtaining academic credits in science, health, math, English, physical education, and technology through the context of the umbrella topics of environmental health and environmental science. Course content is presented in thematic units through which students make connections to environmental health and traditional academic subjects. The 9th grade component consists of five units including Biodiversity, Endangered Species, Air Quality, Land, and Water.
Fun Environmental Health Activities: A Web-Based Resource for K-12 Teachers.
S D Hines, J L King. Southwest Environmental Health Sciences Center, University of Arizona, Tucson, AZ, USA. The Community Outreach and Education Program (COEP) at the Southwest Environmental Health Sciences Center is continually compiling and creating engaging, high quality environmental health K-12 classroom activities. The web is a powerful tool to easily disseminate these materials to interested teachers. To more fully utilize the Internet, the COEP has developed a website and promotional materials called "Fun Environmental Health Activities" where teachers can access web-based materials developed by COEP or download lectures and curricula. This poster shows the breadth of materials and resources available to teachers through this website.
Microbial Population Dynamics During 3-Chlorobenzoate Degradation in Soil.
TJ Gentry, DT Newby, KL Josephson, and IL Pepper. Department of Soil, Water, and Environmental Science, University of Arizona, Tucson, AZ, USA. Changes in microbial populations during degradation of 3-chlorobenzoate (3-CB) in soil were evaluated in a laboratory study. Madera sandy loam was amended with 0, 500, or 1000 μg 3-CB/g dry soil. Selected microcosms were inoculated with the 3-CB degrader Comamonas testosteroni BR60. Degraders were enumerated on Medium A containing 3-CB. Isolated degraders were grouped for identification based on enterobacterial repetitive intergenic consensus sequence-PCR (ERIC) fingerprints. In the uninoculated microcosms, degraders increased from undetectable levels to 6.6 x 107CFU/g in the 500 μg 3-CB/g microcosms but no degraders were detected in the 1000 μg 3-CB/g samples. In both the 500 and 1000 μg 3-CB/g inoculated microcosms, degraders increased from the initial inoculum of 1.0 x 106 CFU/g to around 1.5 x 108 CFU/g, and then decreased following degradation of 3-CB. All degraders isolated from the uninoculated microcosms produced one of two unique ERIC fingerprints, whereas all degraders from the inoculated soil had fingerprints identical to those of C. testosteroni. Bioaugmentation increased the rate of 3-CB degradation. However, after 18 d when selected microcosms were re-amended with 500 μg 3-CB/g, degradation was more rapid in the uninoculated microcosms. The results indicate that bioaugmentation may increase the rate of 3-CB degradation, but inhibit the development of indigenous populations that may ultimately be more effective degraders in the soil environment.
Streamlining Gene Bioaugmetation: Determining the Relative Importance of Growth Rates vs. Gene Transfer Rates.
C J Haney. Department of Microbiology and Immunology. Soils co-contaminated with both organic and metal pollutants are difficult to bioremediate via conventional means due to the high mortality rate of the introduced remediative organism as a result of the dual stresses. To get around this problem, it may be possible to transfer the genes necessary for bioremediation, via conjugation, to the indigenous soil population, a technique known as gene bioaugmentation. Using a model system in vitro, the rate at which the resultant transconjugant population grows will be analyzed to determine whether the growth rate is more dependent upon growth of the individual organisms, multiple gene transfer events by the introduced donor, or gene transfer from transconjugants to indigenous organisms. To do this, a novel donor system will be engineered using Ralstonia eutropha JMP134 and it's plasmid, pJP4, which codes for both mercury resistance and degradation of the organic pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). A "kill gene" (Gef) will be inserted into the JMP134 chromosome, making it counterselectable, and green fluorescent protein (GFP) will be inserted into the plasmid pJP4, allowing for rapid detection of transconjugants. Gene transfer rates can then be compared between the donor and recipient by using isolated transconjugants as donors in separate experiments. The importance of organismal growth rates in population growth rates will then be assessed by comparison with pure culture growth rates. This information will provide information as to how bioremediation via gene bioaugmentation can be better facilitated.
Application of a Biodegradation and Transport Model Incorporating Microbial Lag to Soil Systems of Increasing Heterogeneity and Biological Diversity.
S K Snyder1, F L Jordan1, L Li1, R M Maier1, and M L Brusseau1,2. Deparment of Soil, Water and Environmental Science, Department of Hydrology and Water Resources, University of Arizona, Tucson, AZ, USA. A biodegradation and transport model incorporating microbial lag was developed, and its predictive capability was examined by comparing simulation results to an array of experimental results obtained from systems of increasing complexity. Salicylate was chosen as the model organic compound because it experiences negligible retardation and is a known intermediate in the biodegradation pathway of many common organic contaminants (such as naphthalene and phenanthrene). The first system consisted of a sterilized, homogeneous quartz sand inoculated with a pure culture of Pseudomonas putida RB1353, a strain of bacteria capable of degrading the model organic compound. The other two systems were soils (of differing textures) containing indigenous populations capable of degrading salicylate. For all systems, batch mineralization studies were performed, using the same population as in the miscible-displacement experiments, to determine biodegradation and bacterial growth kinetic parameters (including lag times). Kinetic parameters determined from batch studies and hydrodynamic parameters obtained from miscible-displacement experiments performed using conservative tracers were utilized in the model to allow independent prediction of the experimental results. Preliminary findings demonstrate that simulation results were significantly different from the experimental results in the absence of the incorporation of cell elution and microbial lag into the model. Additionally, cell elution, substrate biodegradation and transport, and bacterial growth demonstrated enhanced variability in the systems with indigenous populations and increased heterogeneity when compared to the single-isolate system.
The Development of an Indigenous Phenanthrene Degrading Community during Long-Term Exposure to Phenanthrene under Saturated Flow Conditions.
A A Bodour, J-M Wang, M L Brusseau, and R M Maier. Department of Soil, Water and Environmental Science, University of Arizona, Tucson, AZ, USA. The widespread environmental contamination by polycyclic aromatic hydrocarbons (PAHs) has led to increased interest in the use of in situ bioremediation as a cleanup strategy. However, few bioremediation studies have investigated indigenous populations and population dynamics following exposure to a PAH. The goal of this study was to examine the temporal response of an indigenous soil population to long term exposure to phenanthrene. A column was packed with an uncontaminated loamy sand soil (organic matter content 2.7%) and saturated conditions were established with 0.005 M CaCl2. Following saturation, the column was exposed to a solution saturated with phenanthrene (~1.2 mg/L). Effluent samples were collected and analyzed for phenanthrene concentration and microbial counts. Counts were performed on mineral salts media + phenanthrene (phenanthrene degraders) and R2A agar (heterotrophic counts). Phenanthrene degraders were separated according to growth rates. Colonies growing within 3 days were called fast growers, while slow growers referred to colonies enumerated on day 14. Unique isolates were chosen and isolated from the fast and slow growers as well as from the soil at the end of the experiment. Isolated microorganisms were then grouped either by ERIC analysis of their genomic DNA or by morphology. For each unique group, 16S rDNA PCR was performed and then sequencing analysis was used to identify the isolates. A total of 24 different phenanthrene degraders were obtained. Of the 24 isolates, only three isolates were found both in the effluent samples and in the soil from the column. These results indicate that attached and free-living microbes that compete for substrate are different. Overall, these results suggest that a diverse population of microbes participated in phenanthrene degradation and further, a succession of populations took place.
Influence of Multiple Bacterial Populations on Phenanthrene Degradation, Bacterial Cell Elution, and Species Distribution.
B M Patterson1, M L Brusseau1,2, R M Maier1, R Frye1. 1Department of Soil Water and Environmental Science, 2Department of Hydrology and Water Resources, The University of Arizona, Tucson, AZ, USA. A single set of degradation coefficients is typically used when representing biodegradation in contaminant transport models. Implicit to this approach is the assumption that only a single degrading isolate exists, or that the entire community of degraders more typically present in natural systems has a uniform, constant growth rate and affinity for the contaminant. This assumption was evaluated through a miscible displacement experiment conducted using a column packed with a soil containing an indigenous microbial community comprised of 24 identified phenanthrene-degrading isolates. Results produced oscillating phenanthrene concentrations in the column effluent, indicating potential competitive interactions among the isolates. A second series of experiments, conducted in a simplified system comprised of sand and 1,2, or 3 indigenous isolates, examined the effects of species interactions on phenanthrene degradation and bacterial cell elution. Bacterial growth rates, density of cells within the column, and bacterial distribution were also evaluated. Results show single bacterial species produced relatively stable cell elution and phenanthrene concentrations in the effluent. Conversely, the behavior in the multiple species systems indicated synergistic and antagonistic interactions occurred among the species. These results illustrate that the dynamics of heterogeneous microbial communities should be considered when evaluating contaminant biodegradation and transport in subsurface systems.
An Ecological Survey of Biosurfactant-Producing Microorganisms in Arid Southwestern Soils.
A A Bodour and R M Maier. Department of Soil, Water and Environmental Science, The University of Arizona, Tucson, AZ, USA. Biosurfactants are a unique class of compounds that have been shown to have a variety of potential applications including; enhanced remediation of organic- and metal-contaminated sites, enhanced transport of bacteria, enhanced oil recovery, cosmetic additives, and biological control. However, little is known about the distribution of biosurfactant-producing microorganisms in the environment. The goal of this study was to examine the ecology of surfactant-producing organisms in order to determine how commonly they occur in pristine and contaminated sites. A series of contaminated (metals or hydrocarbons) and uncontaminated soils were collected. Soils were plated on R2A agar and each unique isolate was grown in mineral salts medium containing 2% glucose to screen for biosurfactant production. Supernatants from each culture were tested using the drop-collapse method to determine their ability to reduce surface tension of a solution. Microorganisms classified as positive for biosurfactant production from the drop collapse test were then grouped by ERIC or REP analysis of their genomic DNA. For each unique group of biosurfactant producers, 16S rDNA sequencing was performed to identify the isolates. A total of 45 biosurfactant-producing isolates were obtained. Of the 45 isolates, the majority were gram positive and were obtained from heavy metal-contaminated (cadmium and/or lead) or uncontaminated soils. Overall, these results indicate that biosurfactant-producing organisms are found in most soils suggesting that in situ production of biosurfactants in contaminated sites may be feasible.
Metals Associated with Aquatic Plants in an Acid-Mining Contaminated Stream: The Role of Metal Plaques.
T L Corley, and M H Conklin. Department of Hydrology and Water Resources, The University of Arizona, Tucson, AZ, USA. Past acid-mining activities near Globe, Arizona released metal contaminants into the perennial reach of Pinal Creek. Dissolved Mn is the dominant metal in Pinal Creek with lower concentrations of Zn, Ni, Cu, Fe, and Co. Concentrations of these metals were determined in samples of water speedwell, rabbitfoot grass, algae and moss collected over a 2.5-year period. Plant concentrations were factors of 100 to over 1,000,000 greater than surface water concentrations. Correlations between plant concentrations and physical and chemical parameters of Pinal Creek were examined. Surface water concentrations correlated with concentrations of all metals except Fe in water speedwell, all metals in rabbitfoot grass, Mn and Zn in algae, and Mn, Co, and Cu in moss. Positive correlations with pH were only found for Zn in rabbitfoot grass and algae, and Mn in water speedwell, algae and moss. More importantly, Mn concentrations showed positive correlations with the concentrations of Zn, Ni, Co, Cu, and Fe in water speedwell and algae, Co in rabbitfoot grass, and Zn, Ni, and Co in moss. The results strongly suggest Mn-containing metal plaques and extracellular precipitates have major roles in determining the total metal concentrations of the Pinal Creek samples.
Reductive Dissolution of MnO2 by Fe(II): Effects of Chemical Gradients and Intermediate Phase Structural Information.
John E. Villinski1, Peggy A. O'Day2, John R. Bargar3 and Martha H. Conklin.1 1Department of Hydrology and Water Resources, The University of Arizona, Tucson, AZ, 2Geology Department, Arizona State University, Tempe, AZ, 3Stanford Synchrotron Radiation Laboratory, PO Box 4349, MS 69, Stanford, CA. An in situ, real-time, synchrotron X-ray absorption spectroscopy study of the reductive dissolution of MnO2 be Fe(II) at pH 3 was performed in a novel flow-through reaction cell at SSRL. While the path length was only 7 mm, different results were obtained from collecting spectra at the inlet (upgradient) and downgradient portions of the bed. Analysis of XAS spectra indicated that Mn(IV) reduction occurred without a detectable presence of a reaction intermediate phase upgradient. Both XANES and pre-edge spectra indicated an intermediate phase containing tetrahedrally-coordinated Mn, and most likely Fe(III), was present downgradient. This is consistent with the observed advection and precipitation of Fe(III). The absorbance of the intermediate phase reached a constant value after 30 minutes of a 2 hour experiment, and thus the intermediate phase appears to exert control over the release of Mn(II) to solution. By probing different regions of a mineral-solution reaction path spectroscopically, a more complete picture of the reactions controlling the reductive dissolution is obtained.
Electrochemical and Spectroscopic Study of Arsenate Removal from Water using Zerovalent Iron Media.
J Farrell1, N T Melitas1, J Wang1, M Conklin2 and P O'Day3. 1Department of Chemical and Environmental Engineering 2Department of Hydrology and Water Resources University of Arizona, Tucson, AZ 85721 3Department of Geological Sciences, Arizona State University, Tempe, AZ 85287. This study investigated the mechanisms involved in removing arsenate from drinking water supplies using zerovalent iron media. Batch experiments utilizing iron wires suspended in anaerobic arsenate solutions were performed to determine arsenate removal rates as a function of the arsenate solution concentration. Corrosion rates of the iron wires were determined using Tafel analysis. The removal kinetics in the batch reactors were best described by a dual rate model in which arsenate removal was pseudo-first order at low concentrations and approached zeroth order in the limit of high arsenate concentrations. This kinetic behavior was attributed to surface saturation effects with increasing arsenate concentration. The presence of arsenate decreased iron corrosion rates. However, constant corrosion rates were attained indicating that the passivation processes had reached steady state. Tafel analysis did not reveal the electrochemical reduction of arsenate whereas arsenate complex formation on the anodic sites of the iron was indicated. Faster arsenate removal was associated with higher corrosion rates and more completely oxidized iron. X-ray absorption spectroscopy analyses indicated that all arsenic associated with the zerovalent iron surfaces was in the +5 oxidation state. Interatomic arsenic-iron distances determined from EXAFS analyses were consistent with bidentate corner-sharing among arsenate tetrahedra and iron octahedra. Results from this study show that under conditions applicable to drinking water treatment, arsenate removal by zerovalent iron media involves surface complexation only, and does not involve reduction to metallic arsenic.
Kinetics of Soluble Chromium Removal from Contaminated Water by Zerovalent Iron Media: Corrosion Inhibition Effects.
N T Melitas, J Farrell and O Chuffe. Department of Chemical and Environmental Engineering University of Arizona, Tucson, AZ 85721. Permeable reactive barriers containing zerovalent iron are being increasingly employed for in situ remediation of groundwater contaminated with redox active metals and chlorinated organic compounds. This research investigated the effect of chromate concentration on its removal from solution by zerovalent iron. Soluble chromate removal by iron wires was measured in batch experiments for initial chromium concentrations ranging from 100 to 10,000 µg/L. Chromate removal was also measured in columns packed with zerovalent iron filings over this same concentration range. Electrochemical measurements were made to determine the free corrosion potential and corrosion rate of the iron reactants. In both the batch and column reactors, chromium removal rates declined with increasing chromate concentration. This indicates that simple first or fractional order kinetic models are not useful for describing chromate removal kinetics. Corrosion current measurements indicated that the rate of iron corrosion decreased with increasing chromium concentrations between 0 and 5,000 µg/L. Analysis of Tafel polarization diagrams indicated that iron corrosion was hindered by both cathodic and anodic inhibition. Cathodic inhibition was indicated by a decrease in the exchange current for the water reduction reaction. Anodic inhibition was attributed to the buildup of Cr(III)/Fe(III) oxides at anodic sites on the iron surfaces. Even at the most passivating concentration of 10,000 µg/L, chromate removal in the column reactors reached a steady state, indicating that the passivation had also reached a steady state. Although chromate contributes to iron surface passivation, the removal rates are still sufficiently fast for iron barriers to be effective for chromate removal at most environmentally relevant concentrations.
Catalytic Hydrodechlorination of Gas-Phase Trichloroethylene on Media Supported Platinum.
O Orbay, X Ju, R G Arnold, E A Betterton, W Ela. Department of Chemical and Environmental Engineering, University of Arizona, Tucson, AZ, USA.Trichloroethylene (TCE) is among the most pervasive contaminants in groundwater and soil due to widespread historical use and improper disposal practices. Development of technically feasible, cost effective technologies for TCE remediation at contaminated sites could save a great deal of money. Dechlorination of gas-phase TCE using elemental hydrogen on a platinum catalyst surface was investigated in this research. Using 5g of 0.5% Pt on an Al2O3 support and 6,423ppmv TCE in N2(g), >99% TCE destruction was obtained at an estimated residence time of 3s at room temperature. Much faster reaction rates were available if the column was heated. Ethane production accounted for more than 90% of the TCE transformed. No partially halogenated intermediates were observed except for chloroethane. The advantage of gas-phase hydrodechlorination over aqueous-phase is in the ease with which H2 can be provided. Mass transfer limitations are also less likely to limit reaction rate and reaction products are, on average, less chlorinated than in similar aqueous-phase systems. Among the problems of this technology is catalyst deactivation. During these experiments, the activity of the catalyst decreased due to sintering, coating, poisoning with HCl, or a combination of these effects. Addition of KOH decreased the observed deactivation rate. The regeneration of catalyst by O2/H2 treatment is under investigation. Due to its apparent simplicity, the process offers promise for destruction of chlorinated solvents in gas streams produced by gas-sparging or soil vapor extraction operations.
Removing TCE by Membrane Air Stripping.
He, T T Nakajima, R G Arnold, W Ela, and E A Betterton. Department of Chemical and Environmental Engineering, University of Arizona, Tucson, AZ, USA. The use of hydrophobic microporous polypropylene hollow fibers for transferring TCE from water to gas phase was evaluated in this study. Contaminated water and (initially) clean air were passed in countercurrent mode through the hollow fiber reactor. Water was on the shell side (external to the hollow fibers). The hydrophobic membrane prevents water and air from mixing but permits the transmembrane transfer of hydrophobic solutes like TCE. Following transfer to the gas phase, such contaminants can be conveniently destroyed using a number of catalytic routes under development in this lab. The membrane air stripping process offers several advantages over conventional packed-tower air stripping, including higher overall mass transfer coefficient (KLa), independence of the transfer surface area from the air or water flow rates, lower required air flow rate, avoidance of mist development, elimination of tall structures, and more efficient off-gas treatment with GAC. A reactor performance model was developed on the basis of empirical correlations, and engineering economics were considered.
Photo-treatment of Perchloroethene in Soil Vapor at Mission Uniform and Linen Service, Tucson, AZ.
BE Barbaris1*, DS Samorano2, EA Betterton1, RG Arnold2, WP Ela2, LJ Berry2, JN Lever1. 1Department of Atmospheric Sciences ( 520-621-6832 ); 2Department of Chemical and Environmental Engineering ( 520-621-6044 ); The University of Arizona, Tucson, AZ 85721. Perchloroethene (PCE) is being scrubbed from contaminated soil vapor by contact with a 2-popanol/acetone solution (9:1 v/v) and then destroyed by exposure to sunlight in a photoreactor. The treatment system (scrubber and photoreactor) has been tried and tested at two landfill locations over the past two years and is currently operating at the Mission Uniform and Linen Supply facility, 301 South Park Avenue in Tucson. Soil vapor is being extracted and treated by contractors using a conventional granulated carbon absorption system. Soil vapor containing ~200 ppmv PCE is readily obtained for our purposes by diverting a small flow of soil vapor (1-10 L/min) to the experimental treatment system. In the experimental system, contaminated soil vapor is pumped at a rate of 10L/min into the bottom of a 60 cm x 5 cm countercurrent column packed with ½" ceramic Berl saddles. PCE is stripped by dissolution in the 2-propanol/acetone mixture that flowed downward through the scrubber. Field data indicate that the scrubbing efficiency is consistently above 95 percent. The 2-propanol/acetone solvent, containing the scrubbed PCE, is pumped to the rooftop photoreactor, a 3-meter glass tube (2.5 cm diameter), where the PCE is destroyed by solar-promoted photolysis (Betterton et al., Environ. Sci. Technol., 2000, 34, 1229 - 1233). The solvent is then returned to the scrubber to close the loop. The solvent is pre-loaded with PCE throughout the night and then exposed to sunlight while being recirculated in the photoreactor during the day (without further contact with the soil vapor). The effectiveness of the photoreactor is determined by comparing liquid-phase concentrations of PCE and other targets in the reactor influent and effluent. PCE concentrations are typically lowered by 90 percent or more within one hour of exposure. Results also indicate that the reaction is inhibited by oxygen in the soil vapor (14%) percent. However, we are able to eliminate (photoreduce) interfering oxygen and also the PCE by increasing the exposure time.
Fate of Endocrine Disrupting Chemicals - During Wastewater Treatment and Polishing Treatments.
Conroy O1, Turney KD1, Lansey KE2, and Arnold RG1. Departments of Chemical and Environmental Engineering1 and Civil Engineering2 University of Arizona, Tucson, AZ. There is widespread concern over environmental and human health effects arising from exposure to estrogenic and other endocrine-disrupting compounds in water. In this study, a competitive binding assay for the human estrogen receptor, ER-b, was used to assess the presence of endocrine-disrupting compounds during wastewater treatment and natural processes that polish wastewater effluents - specifically, infiltration to groundwater and passage through a constructed wetlands. Samples were taken from the Roger Road Wastewater Treatment Plant, the Sweetwater Recharge Facility, and the Constructed Ecosystem Research Facility in Tucson, AZ. Organic constituents were concentrated on C18 disks for determination of an EC50 value, the concentration that displaced 50% of the labeled estrogen from ER-b. EC50 increased at least three-fold as a consequence of secondary (bio-tower) wastewater treatment. However, a 35-fold increase in the EC50 was observed during the infiltration of secondary effluent to groundwater at 130 feet below land surface. A decrease in the EC50 (greater endocrine-disrupting potential) resulted from wetlands treatment.
Up-Regulation of P68 and GDI in Rabbit Renal Cortical Slices Exposed to Low Level Arsenic.
X-H Zheng and A. J. Gandolfi. Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ Our previous studies demonstrated rabbit renal cortical slices can be used to study the low-level toxic effects of arsenic compounds [As(III) and As(V)]. Using cDNA microarray, we have determined the gene expression profile in the precision-cut rabbit renal cortical slices exposed to low-level As(III) and As(V). Although there were common genes affect by both As(III) and As(V), there were also specific gene only altered by a specific As chemical species. Among the differential expressed genes, p68 (the DEAD box protein, which is an established RNA-dependent ATPase and RNA helicase), is up-regulated in As(III)-treated slices. GDP-dissociation inhibitor (GDI) is also up-regulated in As(V)-treated slices. To validate the microarray data, cDNA clones of p68 and GDI were isolated, and the sequences of the cDNA clones were confirmed prior to purification and probe labeling. Adult New Zealand White rabbit renal cortical slices were incubated with (10 nM - 10 m M) As(III) or As(V) in waymouths 752/1 media. At various time points (2-8 hr) total RNA was isolated from 2 pooled rabbit renal cortical slices, separated on 1.2 % agarase/formaldehyde gel, and transferred to nylon membrane. The blots were probed by (a-32P) dCTP labeled cDNA probes specific for p68 or GDI. The Northern Analysis revealed up-regulation of p68 in As(III)-treated slices (- 2-fold from control) that was seen as early as 2 hr. Whereas the up-regulation of GDI in As(V)-treated slices increased 40 % by 6 hr. Our findings are in agreement with our earlier cDNA microarrays studies and help us identify specific marker genes that are indicative of a low-level exposure to specific chemical forms of arsenic. [NIH ES 04940]
Low-Level Arsenite does not Affect the Ubiquitin Conjugating Activity of Rabbit Renal Slices or Human Embryonic Kidney Cells.
D S Kirkpatrick, J M Catania, KV Dale, and A J Gandolfi.Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ, USA.Our previous work with rabbit renal slices has shown that low-level arsenite exposures can stimulate an accumulation of ubiquitin (Ub)-protein conjugates. Additionallly, arsenite exposure (1 m M, 24 hr) in human embryonic kidney (HEK) cells stimulated a similar increase in the amount of Ub-conjugated proteins. Increases were detected in HEK lysates by western blotting with anti-ubiquitin antibodies. It was hypothesized that the increases in Ub-protein conjugates seen by western blot result from an arsenic-induced increase in the levels/activity of the Ub-conjugating enzymes. To investigate this mechanism, rabbit renal slices and HEK cells were exposed to nM-m M concentrations of arsenite. Lysates were tested for their ability to incorporate 125I -labeled ubiquitin into ubiquitin-protein conjugates. Conjugation of 125I-ubiquitin to cellular proteins is an ATP-dependent process carried out by a family of ubiquitin-conjugating enzymes. Labeled samples were separated by SDS-PAGE and detected by autoradiography. Bands in the gel corresponding to a molecular weight of greater than 40 kDa were determined to be cellular protein conjugated by ubiquitin. Data from these experiments show no significant differences in the amount of protein-bound 125I-Ub between control slices and those treated with up to 5 m M arsenite. Similarly, data from HEK cells treated with concentrations as high as 1 m M arsenite showed no changes in the ability to incorporate 125I-Ub. Since these data discount the importance of arsenic-induced increases in conjugating activity, previously noted increases in conjugated proteins must be resulting from alternate mechanisms. Among the possible alternatives are alterations in proteosome activity or effects on expression of ubiquitin itself. Ongoing work seeks to identify this mechanism as well as the specific proteins conjugated by ubiquitin following low-level arsenic exposure. (NIH ES04946, NIH ES 06694, NIH ES 07091)
Monomethylarsonous Acid (MMAIII) and Dimethylarsinous Acid (DMAIII) Toxicity in Hamsters.
JS Petrick1, WR Cullen3, HV Aposhian1,2. Department of Pharmacology & Toxicology1 and Molecular & Cellular Biology2, The University of Arizona, Tucson, AZ; Department of Chemistry3, University of British Columbia, Vancouver, BC, Canada. Recent toxicological studies have implicated trivalent methylated arsenicals as toxic intermediates of inorganic arsenic metabolism (Petrick et al., 2000). Monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII) are methylated trivalent arsenicals produced in the methylation of inorganic arsenic. MMAIII is significantly more cytotoxic than arsenite in mammalian cell culture. Cytotoxicity of DMAIII was approximately equal to that of sodium arsenite (Styblo et al., 1999). Arsenite is a potent inhibitor of mammalian pyruvate dehydrogenase (PDH) in vitro. The inhibition of this critical mitochondrial enzyme by arsenite, MMAIII or DMAIIIwas studied in vitro in kidney homogenate of untreated male Golden Syrian hamsters. Arsenite of MMAIII [as CH3AsI2 or as (CH3AsO)4] inhibited PDH activity in vitro by 50% at concentrations of 136, 69, and 65 m M, respectively. DMAIII, as (CH3)2AsI, did not inhibit PDH at concentrations up to 400 m M. MMAIII is thus a two fold more potent inhibitor of hamster kidney PDH in vitro than sodium arsenite. These findings implicate vicinal dithiol (lipoic acid) binding as a potential mechanism of action for MMAIII and show that MMAIII is a toxic intermediate of inorganic arsenic metabolism. (Supported in part by the NIEHS Superfund Basic Research Program Grant ES-04940 and SWEHSC P30-ES-06694).
Sodium Arsenite Enhances AP-1 And NFk B DNA Binding and Induces Stress Protein Expression in Precision-Cut Rat Lung Slices.
J B Wijeweera and R C Lantz. Department of Cell Biology and Anatomy, University of Arizona, Tucson, AZ, USA. Arsenic is a known human carcinogen. These studies were designed to examine the impact of low arsenite concentrations on immediate early gene expression in precision-cut rat lung slices. Precision-cut lung slices are a versatile in-vitro system for toxicity studies as they preserve the architecture and cellular heterogeneity of the lung. Since 0.1-100 m M arsenite did not compromise slice viability at 4 hr, effects of arsenite on the expression of c-jun/AP-1, NFk B, HSP 32, HSP 72, HSP 60 and HSP 90 were studied using these concentrations of arsenite at 4 hr. Nuclear c-jun was increased by 10 m M and 100 m M arsenite, while NFk B was not affected. Gel shift assays indicated that 10 m M arsenite resulted in an enhanced DNA binding activity of both AP-1 and NFk B. Confocal microscopic analysis of AP-1 indicated nuclear localization of this transcription factor mainly in type II epithelial cells and alveolar macrophages. Nuclear localization of NFk B was lower than that observed for AP-1 while, most of the NFk B was localized to cytoplasm of type II epithelial cells and alveolar macrophages. HSP 32 was increased by 1.0 m M and 10 m M arsenite while, HSP 72 was increased by only 100 m M arsenite. HSP 60 and HSP 90 were not changed by arsenite. These studies indicate that non-cytotoxic concentrations of arsenite are capable of affecting signal transduction pathways and gene expression in the lung.
The Influence of Previous Arsenic Exposure on the Rate of Arsenite Oxidation by Soil Microorganisms.
EA Casarez1, JB McQuaid2, IL Pepper2, and DE Carter1. 1Department of Pharmacology and Toxicology; 2Department of Soil, Water, and Environmental Sciences; University of Arizona, Tucson, AZ, USA. Arsenic (As) is ubiquitous in the environment, both as a natural component of rocks and minerals and as a contaminant from human activities. Arsenic may be present in different forms, each differing in bioavailability, mobility, and toxicity. The species of As present is affected by both biotic and abiotic influences. This research compares the rates of arsenite (AsIII) oxidation in soils with different histories of As contamination. We hypothesize that previous exposure to As has affected the soil microbial population, which will therefore change how additional As is metabolized. A control soil (no detectable As) and three soils from a smelter site with low, medium, and high total As levels were spiked with 0, 100, 500, or 1000ppm As as AsIII. The soils were monitored for microbial growth and water extractable As at 0, 1, 3, 7, and 14 days. Arsenic speciation was determined in the water extracts by high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry, while changes in the microbial community were monitored by heterotrophic plate counts and 16S rDNA denaturing gradient gel electrophoresis. The initial number and diversity of the soil microorganisms was less in the soils with histories of higher As contamination. These soils, however, had faster rates of AsIII disappearance, with a concomitant increase in arsenate, than soils previously exposed to low or no As. Trends indicate that the larger the additional AsIII spike, the faster the rate of AsIII disappearance in pre-exposed soils, while the opposite is true in the control soil. The decreases in water-extractable AsIII are biologically mediated since AsIII levels in similarly treated sterilized soil samples remained constant. The additional AsIII had no adverse effects on the number or diversity of microorganisms in the pre-exposed soils after 14d-one microbial population even became more dominant with increasing AsIII. Although AsIII oxidation is seen in all the biologically active soils, the rates of the reaction are correlated with previous As exposure. This may be due to selection pressures for microorganisms with As resistance and AsIII oxidase activity.
Arsenite Cytotoxicity in HK-2 Cells, an Immortalized Human Proximal Tubular Epithelial Cell Line.
MA Peraza, AJ Gandolfi, and DE Carter. Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ. Arsenic is an environmental toxicant and a human carcinogen of the skin, lung, urinary bladder, liver, and kidney. The kidney is a known target organ of arsenic toxicity and is critical for both in vivo arsenic biotransformation and elimination. This study investigates the potential of an immortalized human proximal tubular epithelial cell line, HK-2, to serve as a representative model for low level exposures of the human kidney to arsenic. To determine the toxicity of arsenic in this cell line, HK-2 cells were exposed to sodium arsenite at concentrations ranging from 100 nM to 1 mM for 24 hours. Acute cytotoxicity was assessed by determining the leakage of lactate dehydrogenase (LDH) and the mitochondrial metabolism of the tetrazolium salt, MTT. Data from these experiments show that significant increases in cytotoxicity did not occur at 24 hours at concentrations ranging from 100 nM to 10 µM arsenite. At 24 hours exposure to 25 µM arsenite caused a release of 40% of total LDH, and maximal release of LDH (100%) was achieved with 50 µM, 100 µM and 1mM arsenite. Similar results for the effect of arsenite on mitochondrial MTT metabolism were observed. Exposure to 10 µM and 25 µM arsenite resulted in a 20% and 75% reduction of MTT metabolism, respectively. A maximal decrease (85%) of MTT metabolism occurred in cells exposed to concentrations of 50 µM and above. Chronic cytotoxicity was assessed by determining leakage of LDH at similar concentrations of arsenite for periods of 48 to 168 hours (7 days). Significant increases in LDH leakage were not observed for cells exposed to 100 nM, 1 µM, or 10 µM arsenite at any time point up to 7 days. By 48 hours, release of 75% of total LDH was observed in cells exposed to arsenite at concentrations of 25 µM, and maximal LDH leakage was observed in cells exposed to concentrations of 50 µM and above. Ongoing work seeks to determine arsenic metabolism in HK-2 cells at the sub-cytotoxic concentrations determined in this study. Current and future results will help determine the value of this cell line in the characterization of the effect of low level arsenic exposure on the human kidney. (T32 ES07091; P42 ES04940).
Health Effects of Hazardous Materials Exposures.
JL Burgess, DF Kovalchick, JF Lymp, KB Kyes, and WO Robertson. College of Public Health, University of Arizona, Tucson, AZ; University of Washington; Washington Poison Center, Seattle, WA. Objective: To study adverse outcomes in individuals exposed to hazardous materials. Methods: Individuals exposed during hazardous materials incidents were contacted to complete a questionnaire within 8-40 days, and medical records were reviewed when available. Logistic regression analysis adjusting for correlation using GEE was used to identify predictors of adverse outcome. Results: From 12/97 to 10/99, 87 incidents were reported. 202 (59%) of 339 subjects with contact information were surveyed. 51 (25%) subjects had persistent medical symptoms. 18 (9%) left work or school for >2 days. Medical records were available for 79 (66%) of 119 subjects evaluated in a health care facility. Medical treatment was reported in 46 (58%) and objective abnormalities in 57 (72%). For persistent symptoms, dermal exposures (OR 3.75, 95% CI 1.47-9.59), ³ 3 alcoholic drinks/week (3.16, 1.13-8.80), and present or past use of psychiatric medications (2.68, 1.15-6.28) were significant predictors. For time loss, being divorced, widowed, or separated (8.36, 1.40-50.1), asthmatic (4.92, 1.29-18.7), or having initial dermal symptoms (6.77, 1.34-34.2) were significant predictors. Of patients with medical records, those with preexisting hypertension (10.6, 1.82-61.3) were more likely to receive medical treatment or have objective medical findings, while those with inhalation exposures (0.17, 0.04-0.78) and those decontaminated at the scene (0.14, 0.03-0.69) were at reduced risk. Conclusions: Both incident and individual patient factors were associated with persistent symptoms, time loss, and medical treatment or objective medical findings.
House Dust and Inorganic Urinary Arsenic Levels in Two Arizona Mining Towns.
TA Hysong, JL Burgess, MK O'Rourke. Department of Community and Environmental Health Practice and Policy, University of Arizona, Tucson, AZ, USA. Residents of copper mining and smelting towns may have increased risk of arsenic exposure due to elevated arsenic levels in environmental media. The influence of arsenic in house dust on urinary biomarker concentrations, however, is incompletely known. To determine the relationship of arsenic in house dust to inorganic urinary arsenic concentrations, a door to door survey was conducted from July to October 1999 in Hayden and Winkelman, Arizona. A total of 122 households participated, eight-five of which provided dust samples. Dust was sieved and analyzed for total arsenic content using x-ray fluorescence. Urine was collected at first morning void from each household member willing to participate and analyzed via HG-AAS for total arsenic. Speciation of arsenic was performed in those samples with total arsenic greater than 10μg/L (N=106). Forty-seven households had both inorganic urinary arsenic and house dust samples. In many cases there was more than one urine sample from each home that provided a dust sample. The generalized estimating equation was used to determine the relationship between urinary and house dust arsenic concentrations, allowing adjustment for the correlation of measurements obtained from the same home. After adjusting for age and gender, only eating seafood in the past three days and smoking contributed significantly to inorganic urinary arsenic concentrations. Arsenic in house dust was not significantly associated with inorganic urinary arsenic in this adult population. Results of this study are consistent with others that have reported no association between house dust and inorganic urinary arsenic concentrations in adult populations.
Threshold Affects of TCE Contaminated Maternal Drinking Water on Fetal Heart Development in the Sprague Dawley Rat.
P D Johnson, S J Goldberg, B V Dawson. Department of Pediatric Cardiology, The University of Arizona, Tucson, Arizona, USA Halogenated hydrocarbons such as trichloroethylene (TCE) are among the most common water supply contaminants in the United States and abroad. Epidemiologic studies have found an association, but not cause and effect relation, between halogenated hydrocarbon contamination and increased incidence of congenital cardiac malformations or other defective birth outcomes. Avian and rat studies demonstrated statistically significant increases in the number of congenital cardiac malformations in those treated with high doses of TCE, either via intrauterine pump or in maternal drinking water, as compared with controls. This study attempts to determine if there is a threshold dose exposure to TCE above which the developing heart is more likely to be affected.Sprague-Dawley rats were randomly placed in test groups and exposed to various concentrations of TCE (2.5 ppb, 250 ppb, 1.5 ppm, 1100 ppm) in drinking water or distilled water (Control group) throughout pregnancy. The percent abnormal hearts in the treated groups ranged from 0 to 10.48%, respectively, with controls having 2.1% abnormal hearts. The data from this study indicate not only that there is a statistically significant probability overall of a dose response to increasing levels of TCE exposure but also that this trend begins to manifests itself at relatively low levels of exposure (i.e. <250ppb). If maternal rats are exposed to these levels during pregnancy an associated increased incidence of cardiac malformations in developing rat fetuses is found.
Handling of the Mercury-Chelator DMPS (2,3-Dimercapto-1-Propane-Sulfonic Acid) by the Human Organic Anion Transporter (hOAT1).
F Islinger1,2, WH Dantzler2, SH Wright2. 1Dept of Physiology, University of Arizona, Tucson, AZ; 2Dept of Physiology, University of Wurzburg.Although DMPS is the preferred clinical antidote for mercury poisoning, the cellular and molecular basis of its efficacy is unknown. Previous studies implicated the "classical organic anion secretory pathway" in the renal secretion of DMPS. The basolateral component of this pathway - hOAT1 - has recently been cloned. We used the Xenopus oocyte expression system to study directly the interaction of DMPS and its mercury chelates with hOAT1. Activity of hOAT1 was assessed by measurement of [3H]PAH uptake (Kt 3.9 ± 1.3 m M). Whereas 100 mM of probenecid and bromsulfopthalein completely blocked hOAT1-mediated PAH uptake, 1 mM tetraethylammonium, taurocholate, penicillin G, and 2,3-dimercaptosuccinate had no effect on PAH transport. Reduced DMPS proved to be a potent inhibitor of PAH uptake (IC50 of 22 ± 8.4 m M). Because circulating DMPS is extensively oxidized, we examined the interaction of oxidized DMPS (DMPSS2-) with hOAT1 and found it to be an effective inhibitor of PAH uptake (IC50 of 60 ± 13.6 m M). Titrating 40 m M DMPSH with HgCl2 lead to a systematic decrease in the inhibition of PAH-Uptake; indeed, 40 mM DMPSH plus 40 mM HgCl2 produced no inhibition. We suggest that hOAT1 supports the entry of both reduced and oxidized DMPS into proximal renal cells, but does not interact with metal chelates of DMPS. (ES-06694).
Determinants of Structural Specificity of the Human Organ Cation Transporter, hOCT1.
DJ Bednarczyk, WH Dantzler, SH. Wright. University of Arizona, Tucson, AZ. A stable line of HeLa cells expressing hOCT1 was used to investigate the structural and chemical characteristics that influence binding of substrate to the transporter. Expression of hOCT1-mediated transport was selected for in antibiotic-resistant cells following transient transfection by monitoring mediated accumulation of the fluorescent cation, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl] trimethylammonium. Uptake of [3H]tetraethylammonium into these cells was linear for five minutes, and the 60 minute accumulation of TEA was 20-fold greater than that observed in wild-type HeLa cells. A series of n-tetraalkylammonium compounds was used to determine the effect of increasing lipophilicity on the binding of substrate to hOCT1. Increasing hydrophobicity (increasing alkyl chain length) was correlated with decreasing values for IC50s: TEA (IC50 of 165 mM; n=3); tetrapropylammonium (20 mM; 3); tetrabutylammonium (6.5 mM; 3); and tetrapentylammonium (1.8 mM; 3). The steric influence of planar, hydrophobic moieties on the binding of substrate to hOCT1 was examined using a series of substituted phenylpyridinium compounds. Inhibitory effectiveness was again associated with increasing lipophilicity of these compounds. Such correlative data may provide information about structural and chemical constraints of the organic cation binding site. (DK58251)
Characterization of Molecular Markers of Trichloroethylene Exposure during Heart Development.
FM Blachere, PA Thorne, JM Collier(1), PD Johnson, RB Runyan(1), and O Selmin(2). Department of Pediatrics, Section of Cardiology,(1) Department of Cell Biology and Anatomy, and (2) Department of Nutritional Sciences, University of Arizona, Tucson, AZ. Trichloroethylene (TCE), a halogenated hydrocarbon, is a major contaminant of groundwater in Tucson, Arizona. Preliminary epidemiological and animal studies established a correlation between fetal heart defects and maternal exposure to TCE. In effort to better understand the effects of TCE on cardiac development, our research has focused on elucidating the molecular mechanisms that mediate embryonic heart development. In this study, we have been examining the effects of TCE exposure on rat embryonic heart in-utero. Utilizing the PCR select subtractive hybridization method (SSH), differential gene expression was observed in TCE-treated embryonic hearts. In particular, a number of genes were found to be negatively regulated, including the GPI-linked protein p137 and the sarcoplasmic reticulum calcium regulatory protein Ca+2 ATPase, as a result of TCE exposure. Down regulation of p137 expression was confirmed by RT-PCR and Western analysis in which protein isolated from heart or embryo exposed to 110ppm TCE was probed with p137 polyclonal antibodies. In addition, the same antibodies were shown to inhibit mesenchymal cell invasion using in-vitro collagen gel assays. Current research is underway to determine how TCE regulates expression of Ca+2 ATPase during embryonic heart development. Concurrent to rat in-utero studies, we have also been examining the effects of TCE exposure on an ATCC rat myocyte cell line (H9c2). RT-PCR data indicate that H9c2 cells express the TCE-metabolizing enzymes CYP2E1 and Alcohol dehydrogenase (ADH), both also present in rat embryo. Further experiments are in progress to evaluate the sensitivity of the H9c2 at the gene expression level to TCE exposure. Future work will use the H9c2 cells as an in-vitro system for transfection and reporter gene studies that will shed light on the molecular mechanisms of TCE-mediated gene regulation. Supported by: grant number P42ES04940 from the National Institute of Environmental Health Sciences, NIH.