Abstract  Alkali eye burns cause serious injuries. Because of the potential serious outcomes, a comparison was made of two emergent eye decontamination solutions: normal saline and Diphoterine. Diphoterine is an amphoteric and hypertonic polyvalent agent for eye/skin chemical splash rinsing. Before the study, in vitro and in vivo tests were performed in order to show the interest of the study. The clinical study was approved by the appropriate human subjects committee. All consecutive cases of alkali ocular burns treated at the University Hospital Center of Fort de France were prospectively collected during a 4-year period. Injuries were classified according to the Roper-Hall classification (Grades 1-4). Regardless of the decontamination product used, the remainder of the therapeutic protocol was standardized according to injury Grade. In vitro tests showed that ammonia penetrates through a semi-permeable membrane when the concentration is 0.35N. In vivo tests showed an absence of edema with more preservation of endothelial cells and decrease of intraocular pH with Diphoterine rinsing. During the clinical study, there were 66 cases of ocular alkali burns. Of these, 28 of patients had a unilateral burn. 48 eyes were decontaminated with normal saline and 56 eyes with Diphoterine. For Grades 1 and 2, the elapsed time to reepithelialization was significantly shorter with Diphoterine decontamination. There were insufficient cases in Grades 3 and 4 for statistical comparison. In this clinical study, the elapsed time to reepithialization was shorter in Grade 1 and 2 burns when Diphoterine was the decontamination solution. These results are comparable to prior observational studies and with in vivo studies. Diphoterine should be considered as an alternative to normal saline or other decontamination solutions for chemical eye splashes.

Abstract  Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in a variety of lesions in mammals including alterations in liver function and lipid metabolism, immune system suppression, endocrine and nervous system dysfunction, and severe skin lesions. The majority of TCDD?s biological effects are mediated through activation of the aryl hydrocarbon receptor (AhR), a member of the bHLH-PAS family. We have chosen to examine the effect of TCDD and the AhR pathway on dermal fibroblasts because they play an integral role in skin homeostasis by producing cytokines and other factors that influence epidermal proliferation and differentiation. Recent data from keratinocytes indicate that TCDD exposure mediates some of its effects through altering genes involved in cellular senescence. We propose that TCDD and the AhR pathway alter cellular proliferation and senescence by altering expression of the inhibitor of DNA binding (Id) family of proteins. The Id proteins are a class of HLH proteins that lack a DNA binding domain and function by forming inactive heterodimers with other HLH proteins. The Id proteins also interact with non-HLH proteins involved in regulation of cell cycle. Our data show that normal human dermal fibroblasts (NHFs) are responsive to TCDD, as demonstrated by induction of (cytochrome p450 1B1) CYP1B1 expression. Further, our data demonstrate that TCDD treatment of NHFs results in significant (75-90%) decrease in expression of Id-1 and -3. TCDD-repression of Id-1 and -3 is independent of de novo protein synthesis; co-treatment with cycloheximide has no effect on TCDD inhibition of Id-1 and -3. Co-treatment with the AhR antagonist anapthoflavone partially blocks inhibition of Id-1 and -3 by TCDD, suggesting that TCDD inhibition of Id-1 and -3 is, at least in part, mediated through the AhR pathway. These data show that the Ids are targets of the AhR pathway, and suggest that TCDD alteration of Id expression may be involved in changes in cell proliferation and senescence in dermal fibroblasts.

Abstract  Personal exposures to naturally-occurring asbestos (NOA) were monitored during simulated sports and play activities (hiking, biking, baseball, soccer, basketball, gardening) at 3 public schools and a park in a Sierra foothill community. Sampling occurred over 11 days during the dry season in El Dorado Hills, CA, where NOA is present in areas of intense residential development. Asbestos levels in personal and ambient air samples were measured as PCME (phase contrast microscopy equivalent) fibers using the ISO 10312 analytical method. Personal asbestos exposures (n=5 for each activity) ranged 0.001 to 0.034 f/cc during simulated activities and were compared to ambient stationary samples (n=5 each day; grand mean = 0.0008 f/cc) collected concurrently nearby in the community. Results: Asbestos was detected in almost all personal exposure and ambient air samples. The predominant fiber type was an amphibole of the actinolite-tremolite solid solution series. Simulated sports and play activities created personal asbestos exposures that ranged 2-fold to 62-fold higher than concurrent ambient air levels in the community. The highest asbestos exposures (12-fold to 62-fold higher than concurrent ambient) occurred to participants on bare, natural dirt in a student garden and on a trail used for hiking and biking. Elevated asbestos exposures during sports activities on grass (soccer) and asphalt (basketball) ranged 3-fold to 16-fold higher than concurrent ambient levels. Conclusions: Sampling breathing zone air during simulated activities is a superior technique for evaluating personal asbestos exposures than is stationary air sampling. Typical sports and play activities can create significantly elevated personal asbestos exposures for children in areas where NOA is present. Since amphibole fibers persist in the body for many years and children can be expected to survive the decades-long latency period associated with asbestos-related diseases, this exposure represents a potential public health issue worthy of attention from all stakeholders.

Abstract  Although microarray technology has emerged as a powerful tool to explore expression levels of thousands of genes or even complete genomes after exposure to toxicants, the functional interpretation of microarray datasets still represents a timeconsuming and challenging task. Gene ontology (GO) and pathway mapping have both been shown to be powerful approaches to generate an unbiased view of biological processes and cellular components impacted by toxicants. However, current methods only allow for comparisons across two experimental settings at one particular time point. In addition, the resulting annotations are presented in extensive gene lists with minimal or limited quantitative information, data that is crucial in the application of genomic data for toxicology and risk assessment. To facilitate quantitative interpretation of dose or time dependent genomic data, we propose to use combined average raw gene expression values (e.g. intensity or ratio) of genes associated with specific functional categories derived from the GO database. We have developed an extended program (GO-Quant) to extract quantitative gene expression values and to calculate the average intensity or ratio for those genes significantly altered by functional gene category based on MAPPFinder results. To demonstrate its application, we have applied this approach to a previously published dose and time dependent toxicogenomic data set. Our results indicate that the above system approach can describe quantitatively the degree in which functional systems change across dose or time course. Additionally, this approach provides a robust measurement to illustrate our results compared to single gene assessments and enables the user to calculate the corresponding ED50 for each specific functional GO term, a necessity for risk assessment.

Abstract  Epidemiological assessments of health outcomes are frequently restricted to available data on ambient pollutant concentrations. This constraint, however, does not require that toxicological studies similarly limit their assessments. Since it is proportional to target tissue dose, total exposure (the product of concentration, ventilation, and exposure duration) of a gaseous pollutant like ozone is a better predictor of acute responses than concentration. For particulate matter (PM), however, neither concentration nor total exposure should be presumed proportional to dose. We reviewed studies reporting inflammatory responses to concentrated ambient particles (CAPs). The constituents, sources, and the toxicity of CAPs are thought to vary regionally and seasonally. Where sufficient information was available, we estimated the dose of CAPs to regions of the lung using a mathematical model. No consistent dose-response function was discernable; however, CAPs size distribution data was absent or incomplete in the majority of studies. The lack of such basic information as the size distribution of CAPs prevents accurate dose estimates and adds to the uncertainty in cross study comparisons. Particle size alone is recognized to effect the location and amount of deposition in the lungs as well as subsequent clearance rates. For example, the dose of 0.1 um particles in the pulmonary region of a resting rat is predicted to be about 3-fold greater than for 1.0 um particles at equal exposure concentrations. Consequently, the 0.1 um particles might appear more toxic by virtue of their higher pulmonary dose. The presentation of CAPs size distribution data should also be carefully considered as the particle size distribution may not be log-normal. Accurate reporting of PM concentration and size distributions to yield improved dose estimates in toxicology studies may reduce uncertainty and improve assessment of regional and seasonal PM health effects.

Abstract  The Bovine Corneal Opacity and Permeability Assay (BCP-P assay) has been accepted by the EU national coordinators at their 28th meeting as an alternative to the Draize rabbit eye irritation test and is used to classify a chemical as irritant (R41). The BCO-P assay may be routinely used for assessment of safety and as a test to classify and label chemicals as ?severely irritating to the eyes?. This organotypic assay is considered acceptable by the national authorities where a positive result in respect to severe irritancy is obtained. In this case no further animal testing is necessary. Normally in the BCO-P assay the bovine eyes are collected as fresh as possible after slaughter from an abattoir. The corneas are isolated shortly and the tests are performed as soon as possible (no longer than 12 hours). In order to optimize the test conditions, we have performed some validation studies to estimate the differences of the test results between applying fresh bovine eyes and using eyes which are preserved in MEDIUM 199 Modified supplemented with Dextran. We have also compared the BCO-P results, in which the corneas from the animals in different ages were used. In the validation studies three positive control substances, 2-ethoxyethanol, imidazol (20%) and benzalkoniumchlorid, have been tested for various treatment times. The validation studies are useful for further optimizing the BCO-P test conditions and increasing the reliability of this method.

Abstract  It has been standard practice to use 2 Liters/day and 70 kilogram body weight (approximately 29 mL/day) as the default intake rate for the derivation of chronic, noncancer drinking water standards. The Minnesota Department of Health (MDH) is currently revising the Minnesota Health Risk Limits Rule (HRL) for Groundwater. As part of the rule revision process the MDH evaluated the relationship between the time point at which an adverse effect was observed in a toxicity studies and the duration of exposure used to derive a HRL. The MDH was concerned that intake rates in young children were substantially higher than the standard default intake rate. The USEPA has defined a chronic period as repeated exposure over more than approximately 10% of a lifetime. The 10% of a lifetime guideline has been used for developing chronic reference values (RfDs) as well as for identifying chronic exposure durations in noncancer risk assessments. For a human, 10% of a lifetime corresponds to about 7 years. A time-weighted average over the first 7 years of life indicates that the 90th to 99th percentile intake rates during this time period are 2 to 4-fold higher than the default intake rate of 29 mL/day. To determine whether the effects that were the basis of chronic RfDs occurred at earlier time periods the MDH evaluated RfDs used for the draft HRLs. The results of this evaluation indicated that only 4 of 77 RfDs used to derive draft HRLs were based on effects that required exposures longer than 10% of a lifetime, data for 7 chemicals were insufficient to determine when adverse effects were first observed, and the remaining RfDs or supportive studies showed that adverse effects resulted from exposures of up to approximately 10% of a lifetime. Chronic drinking water standards must address potential adverse health effects from short-term as well as long-term exposures to contaminants in drinking water. The MDH recommends utilizing 10% of a lifetime as a minimal chronic exposure period. The MDH selected the period with the highest intake rate, the first 7 years of life, for use in developing chronic HRLs for noncancer effects.

Abstract  Large granular lymphocyte leukemia (LGLL) has a high and variable spontaneous incidence in aging F344 rats. The current status of knowledge of rat LGLL, including a search for mechanistic data and correlations to human leukemia, was examined so as to develop specific recommendations to improve the evaluation of the LGLL endpoint in bioassays and the appropriateness of using this endpoint in risk assessments (RA). The increasing background incidence of F344 rat LGLL over time and its modulation by extraneous factors (i.e. corn oil gavage) are considered to be key confounders in the identification of treatment related effects in the F344 rat model. Currently, a mode of action cannot be proposed for chemicals that might increase the incidence of LGLL in the F344 rat because there is virtually no mechanistic data available. Some similarity between rat LGLL and a rare human natural killer cell (NK)-LGLL exist, invalidating the historical claims that the rat disease has no human counterpart. However, underlying mechanisms may be different. Because of these concerns, it is recommended that any statistical analysis of LGLL use a more stringent level of significance than the traditional p and lt;0.05 (e.g., p and lt;0.01) as an indicator of possible carcinogenic effects. Additionally, specific recommendations and guidance are provided to strengthen the interpretation of possible elevated rates of F344 rat LGLL using an overall weight of evidence approach. Furthermore, consideration of factors such as dose-response, reduction in latency time, appropriate historical controls, neoplasia at other sites, reproducibility in another strain or species the weight of evidence for genotoxicity, toxicokinetics and possible human exposure should all be part of a comprehensive RA for any chemical that might cause increased F344 rat LGLL.

Abstract  Recently, the ICCVAM evaluated four in vitro test methods for their ability to identify ocular corrosives/severe irritants. Ideally, these evaluations would have included an assessment of the ability of the Draize rabbit eye test to correctly identify human ocular corrosives/severe irritants, but comparative human and rabbit data are not available. Therefore, using an in vivo rabbit eye test database of 181 studies, the estimated likelihood of underpredicting a corrosive/severely irritating response as a nonsevere response was determined to evaluate the performance of the Draize test according to the UN Globally Harmonized ocular hazard classification system. The distribution of individual rabbit responses within each severity class was used to estimate the likelihood of under- and/or over-classification for a sequential 1 to 3 rabbit testing strategy. Based on three assumptions about the variability in individual rabbit responses among substances within each classification category, the estimated likelihood for underclassifying corrosives/severe irritants as nonsevere irritants or nonirritants ranged from 4% to 13%. Analyses based on test substance physical form suggested that the underclassification likelihoods for solids were lower than liquids (2.9%-8.3% vs. 5.4%-15.8%, respectively), although these differences were not statistically significant. Estimated likelihoods for underclassification were higher when corrosive/severe irritant classification was based solely on persistent lesions present at observation day 21. By chemical class, carboxylic acids had the highest underclassification likelihood (16.64%). Likelihoods for overclassification of substances as corrosive/severe irritants, based on 596 studies, were estimated to be 7%-8% for Category 2A, 1% for Category 2B, and 0% for nonirritant substances. The overall likelihood of overclassifying a nonsevere/nonirritant, based on prevalence, was and lt;1%.

Abstract  Mouse prostate development starts as projections (buds) of UGS basal epithelial cells into the surrounding mesenchyme on Gestation Day (GD) 16. Budding is thought to be androgen dependent; normal female USG has only rudimentary ventral buds (VBs). In males, in utero TCDD exposure blocks VB formation and reduces dorsolateral bud (DLB) number. To determine if rudimentary VBs in females provide an alternative model to elucidate mechanisms by which TCDD prevents VB formation in males, C57Bl/6J dams were implanted on GD 13 with sustainedrelease pellets containing 5?-dihydrotestosterone (DHT), flutamide, or vehicle. Budding was evaluated by light microscopy on GD 19. DHT-exposed female UGSs closely resembled normal male UGSs. Flutamide-exposed UGSs had the typical female phenotype: no DLBs and rudimentary VBs. Consequently, while DLB bud formation and VB growth are androgen dependent, VB initiation is apparently androgen independent. To determine if rudimentary VBs are capable of developing into VP, GD 19 UGSs were transplanted into the subrenal capsule space of adult male nude mice. After 28 days, tissues were evaluated for prostatic differentiation. Regardless of whether VBs on GD19 were well-developed (male/vehicle, male/DHT, female/DHT groups) or rudimentary (female/vehicle, male/flutamide, female/flutamide groups), all tissues had comparable amounts of mRNA for MP25, a VP-specific secretory protein. These results indicate that rudimentary VBs can develop into fully differentiated VP if androgens are present. Other recent findings demonstrate that TCDD blocks female VB formation in an androgen-independent fashion. The absence of DLBs from female UGS, the sensitivity of female VBs to TCDD, the potential for rudimentary VBs in females to develop into VP, and the ability to control androgen stimulation demonstrate that transplant studies with female UGS provide a useful alternative model to investigate effects of TCDD on VP development.

Abstract  Substitution refers to the preventive measure of replacing a highly toxic substance by a less toxic substance. Based on the consideration of several occupational health, safety and environmental criteria, solvents can be ranked for suggesting the appropriate substitute, for a particular use situation. The present study was undertaken to evaluate the use of several risk ranking algorithms in determining solvent substitution in occupational settings. Fourteen solvents were selected for this study: (a) eight ?traditional? solvents (methanol, trichloroethylene, n-hexane, toluene, dichloromethane, perchloroethylene, acetone and 2-ethoxyethanol) for which substitution is sought; and (b) six ?new? solvents (N-methyl-2-pyrrolidone, dimethylsulfoxide, d-limonene, ethyl lactate, 1-bromopropane and benzyl alcohol) which have been judged after a thorough toxicological and environmental evaluation as acceptable replacements in most situations, except 1-bromopropane. The following 4 methods were applied to the 14 solvents: vapour hazard ratio (VHR), substitution factor index (SUBFAC, Denmark), Air index from the Waste Reduction Algorithm (WAR) of the U.S.EPA, and Indiana Relative Chemical Hazard Score (IRCHS) index. VHR and SUBFAC are simple algorithms that take into account the OEL (Occupational exposure limit) and vapour pressure or evaporation rate to estimate risks. IRCHS is a complex algorithm with several parameters like carcinogenicity, OEL, vapour pressure, flammability and environmental considerations. The Air index is a complex algorithm resulting in an environmental hazard index. According to VHR and SUBFAC, the five solvents judged to be acceptable obtained the best scores. With the Air index classification, d-limonene is ranked worse than 2-ethoxyethanol, while IRCHS classifies 1-bromopropane among acceptable solvents. Overall, while VHR and SUBFAC appear to be the most promising tools on this set of substances, they are limited by not taking environmental factors into account. Better tools need to be developed which integrate occupational health, safety and environmental criteria.

Abstract  Coplanar polybrominated biphenyls (PBBs) and PCBs bind the aryl hydrocarbon receptor(AHR) while non-coplanar congeners do not. Previous work in our lab compared coplanar 3,3?,4,4?,5,5?-hexabromobiphenyl (cHBB) with non-coplanar 2,2?,4,4?,6,6?-hexabromobiphenyl (ncHBB) given on gestational day 5(GD5) to C57BL/6J(B6) mice having the high-affinity AHR and to B6.D2-Ahrd congenic mice with a low-affinity AHR on a B6 genetic background. Neonatal lethality and atrophy of the spleen and thymus occurred in B6 pups whose mothers had received as low 2.5 mg/kg cHBB. No lethality was seen in B6.D2Ahrd or Ahr(-/-) pups whose mothers received 100 mg/kg cHBB or in any pups whose mothers received 100 mg/kg ncHBB. To determine if persistent AHR activation during gestation could explain this phenomenon, we cross-fostered pups from corn oil-treated dams to cHBB-treated dams and pups from cHBB-treated dams to corn oil-treated dams. Pups from corn oil-treated dams all survived the first week postpartum. Only 21% of pups exposed to cHBB in utero could be rescued by cross-fostering to corn oiltreated dams. Interestingly, B6 pups nursed by cHBB-treated dams eventually began losing weight and died by postnatal day 15. There was no lethality in Ahr(-/- ) or B6.D2-Ahrd pups exposed to cHBB in mother?s milk. To examine the distribution of toxicant, we spiked CHBB with 14C-labeled hexachlorobiphenyl and dosed B6 and B6.D2-Ahrd dams on GD5. The radiolabel was 2.2-fold higher in B6 maternal liver and 4.5-fold higher in B6 fetal liver at GD18 compared with B6.D2- Ahrd dams and pups respectively. Our results suggest that persistent activation of the AHR by cHBB is likely the critical factor responsible for the neonatal lethality.

Abstract  The ACI rat is uniquely susceptible to mammary carcinogenesis by estrogen without prior chemical carcinogen initiation and an appropriate animal model for studying human breast cancer. This study was designed to analyze the differential expression of proteins from ACI rat mammary gland and tumor tissues and to identify potential biomarkers for breast cancer diagnosis. The cytosol of normal and tumor mammary gland tissues were collected after continuous 17B-estradiol for 38-46 weeks. Using 24 cm immobiline gel strips for Isoelectric focusing, 500 ug of proteins were focused on each gel, followed by 12% SDS-PAGE. Both samples were in triplicate. Two-dimensional gel patterns were analyzed with Non-Linear Dynamics? PhoretixTM 2D Evolution software 2005. Seventy spots were significantly different between normal and tumor mammary gland tissues. All protein identifications were obtained by LC-MS/MS and TurboSEQUEST database search after trypsin in-gel digestion. Twenty six proteins were identified, 12 of 26 proteins have been associated with carcinogensis of the breast and other tissues based on literature: glutamate dehydrogenase, chloride intracellular channel 1, cytokeratin 19 and nucleolar phosphoprotein B23.1 are up-regulated with tumor development; whereas transaldolase 1, beta-galactoside-binding lectin, mammary growth derived inhibitor, GDP-dissociation inhibitor, glycosylation dependent cell adhesion molecule 1, Annexin V, tropomyosin 1 and calreticulin were down-regulated. Cytokeratin 19 has been suggested as a marker in breast cancer while chloride intracellular channel 1 as a marker in colorectal cancer. MDGI has been reported as a growth inhibitor for beast cancer, since its high protein expression in normal mammary gland tissue vs. very low presence in mammary gland tumor tissue on 2D gels, it can be considered as a bio-marker for mammary carcinogensis. This is the first protein expression analysis of ACI rat mammary gland compared to spontaneous tumors arising in the mammary glands following continuous estrogen exposure.

Abstract  Dienaldehydes are by-products of peroxidation of polyunsaturated lipids and commonly found in many foods or food-products. Trans, trans-2,4-decadienal (tt- DDE), a specific type of dienaldehydes, is abundant in heated oils, and has been associated with lung adenocarcinoma development in women due to their exposure to oil fumes during cooking . Acute exposure to high doses (5 and 10 uM) of tt- DDE significantly increased intracellular oxidative stress (ROS), reduced GSH/GSSG (glutathione status) and decreased cell viability in human bronchial epithelial cells BEAS-2B. However, long-term exposure to non-cytotoxic doses (0.1 and 1 uM) of tt-DDE significantly increased cell proliferation on day 45. At the same time, treatment with 1 uM tt-DDE increased the expression and release of pro-inflammatory cytokines TNFalpha and IL-1beta, but reduced p27 expression. Cotreatment of BEAS-2B cells with either antioxidant N-acetylcysteine or vitamin C prevented tt-DDE induced cell proliferation, release of cytokines and reduction of p27 expression. Therefore, these results suggest that tt-DDE-induced changes may be due to increased ROS production and enhanced oxidative stress. Furthermore, reduction of p27 expression may involve in tt-DDE-induced cell proliferation. Since increased cell proliferation and the release of TNF-alpha and IL-1beta are believed to be involved in tumor promotion, our results suggest that tt-DDE may play a role in cancer promotion.

Abstract  Amino- and nitroderivatives of benzene are widely used in the chemical industry as intermediate products in the production of dyes, medicines, pesticides, etc. For providing safety to people, which have contact with the amino- and nitroderivatives of benzene, it is necessary to study not only the acute and chronic toxicity of these substances, but also the long-term effects, for example embryotoxic effect. Our own studies, and studies of other authors, showed that under conditions of inhalation exposure for 4 hr/day 7 days/week throughout gestation of the Wistar rats such compounds as 2-nitro-4-bromophenol, the 3-nitrobenzoic acid, 3,4-difluoronitrobenzene, 3,4-difluoroaniline, ortho-phenylene diamine, 2,4- dinitroaniline, nitrobenzene, 2,4-dichloronitrobenzene and some others expressed an embryotoxic effect. This effect appeared as increased embryo losses, teratogenicity, changes in the central nervous system, the cardiovascular system, and changes in the physical development of progeny. In this case the Lowest Observed Adverse Levels (LOAELs) for embryotoxic effect were established at the lower levels than the LOAELs for maternal toxicity in pregnant dams. On the basis of using the Quantitative Structure-Activity Relationship (QSAR) analysis, the equations for prediction of both the LOAELs for maternal toxicity and for embryotoxic effects of amino- and nitroderivatives of benzene were obtained. This gives a possibility of approximate determination of the toxic effect of amino- and nitroderivatives of benzene (including embryotoxicity) at the stage of compound rational design even before its synthesis. Thus, in our opinion the occupational amino- and nitroderivatives of benzene may be potent embryotoxic agents, which is necessary to consider during the evaluation of permissible levels of exposure.

Abstract  The risk of abnormal embryonic/fetal development associated with prenatal exposure to polybrominated diphenyl ethers (PBDEs) has not been well characterized. Of the commercial mixtures, penta-BDE mixtures exhibit the broadest range of toxicity. In rats fed standard laboratory diets, penta-BDE exposures up to 200 mg/kg/day (mkd) were not developmentally toxic (reviewed in Darnerud 2003). Research by our group and others has shown that the teratogenicity of some toxicants can be modulated by altering select micronutrient concentrations in the maternal diet. The micronutrient vitamin A is a key regulator of vertebrate embryonic and fetal development. Penta-BDE exposure can disrupt vitamin A homeostasis, thus we speculated that maternal vitamin A status would modulate the teratogenicity of PBDEs. In the current study, we tested the hypothesis that gestational exposure to DE-71 (a penta-BDE mixture) in rats marginal in vitamin A would result in abnormal development. Female Sprague Dawley rats of sufficient or marginal vitamin A status were mated with untreated males. Initiating on gestation day (GD) 6 and continuing daily through either GD 11.5 or 19.5, subsets of rats from each dietary group were administered corn oil (vehicle control) or 18, 60, or 120 mkd DE- 71 by oral gavage. Maternal, embryonic and fetal samples were collected on GD 12.5 and 20 for retinoid measurements and litters were evaluated for gross abnormalities and skeletal development. DE-71 exposure decreased maternal plasma vitamin A concentrations in a dose-dependant manner with the percentage decrease greatest in marginal vitamin A status dams. Conceptal vitamin A metabolism and signaling are currently being characterized. Decreases in crown rump lengths and altered bone formation/ossification were evident in fetuses of marginal vitamin A dams exposed to 120 mkd DE-71. We conclude that a marginal vitamin A status predisposes the conceptus to penta-BDE-induced developmental toxicity and suggest that in women marginal in vitamin A, the reproductive risks associated with PBDE exposure are elevated.

Abstract  Low fetal weight is a sensitive indicator of developmental toxicity in animal studies. While low birth weight may be permanent or transitory, the long-term effects of low birth weight on adult health have not been elucidated. Previous research has shown in humans an inverse relationship between birth weight and risk of adult diseases such as coronary heart disease, hypertension, and diabetes. In this study, pregnant Sprague-Dawley rats were exposed to perfluorooctane sulfonate (PFOS, 18.75 mg/kg, GD 2-6), dexamethasone (DEX, 0.2 mg/kg, GD 14-20), atrazine (ATR 125 mg/kg or 200 mg/kg, GD 14-20), or undernutrition (UNUT, 50% of normal rat diet GD 2-21). Controls were dosed with appropriate vehicle. At birth, all pups were cross fostered to untreated dams. All prenatally exposed pups had significantly lower birth weights than controls, and this persisted through postnatal day (PD) 21. By PD 112, body weights of prenatally exposed rats were not significantly different from control rats. Two indicators of adult diseases are blood pressure and serum glucose level. On PD 56, tail blood pressure was measured. Systolic blood pressure was significantly elevated in male offspring of dams exposed to PFOS (13% increase) or UNUT (27% increase). On PD 70 and PD 112, fasting serum samples were collected and rats were given a 1 g/kg D-glucose challenge. Serial serum samples were collected over 2 hrs, and no significant differences between the prenatally exposed rats and controls were observed. In conclusion, we have found that low birth weight was associated with elevated blood pressure for some, but not all adverse prenatal environments.

Abstract  1,3-Butadiene (BD) is metabolized by cytochrome P450 2E1 to several epoxides that are considered toxic and carcinogenic. The first step of BD metabolism is oxidation to 1,2-Epoxy-3-butene (EB). It has been shown for other P450s that they can be inactivated by covalent binding of reactive metabolites to protein or heme. In this study, potential binding sites of EB in human P450 2E1 were identified to evaluate whether EB binds to residues important for enzyme activity. Therefore, human P450 2E1 was reacted with EB, digested with trypsin and the resulting peptides were analyzed by MALDI-MS. The identity of EB modified peptides was confirmed by tandem mass spectrometry sequencing. These experiments shown that EB binds to four histidine and two tyrosine residues. All modification sites were assigned by at least two adjacent and a minimum of 8 peptide specific fragments. Protein modeling revealed that two of these covalent modifications (His109, His370) are clearly associated with active site, and that their C alpha atoms are located less than 9 A from a known inhibitor binding site. In addition, the side chain of His370 is within 4 A of the heme group and its modification is expected to influence the orientation of the heme in the active site. The C alpha atom of Tyr71 is within 14 A of an inhibitor binding site and within 7 A of the flap undergoing conformational change upon ligand binding, placing Tyr71 near the substrate as it enters and leaves the active site. Several in vitro binding sites of EB in human P450 2E1 were identified. To increase the specificity towards BD we are currently investigating reactions of P450 2E1 with BD in the presence of NADPH for EB binding sites. The data support the hypothesis that EB can inactivate P450 2E1 by covalent modifications and thus add an additional regulatory factor of BD metabolism.

Abstract  Breast cancer is one of the leading causes of premature death in North American women, and the formation and growth of breast tumors are estrogen-dependent. The development of rational treatment strategies for mammary cancer is dependent on understanding regulation of mammary cancer development and growth, and inhibition of these responses. Evidence has shown that cell development and growth are involved in cell communication. Gap junctions are an example of one type of cell communication and can be regulated through PKC signaling pathway. Many hydrocarbons and organochlorine compounds are endocrine disruptors that have been shown to downregulate gap junctions. The regulation of gap junctions by the hydrocarbon, 2,3,7,8-TCDD in breast cancer cells is unknown. The goal of our studies is to determine the effect of TCDD on gap junctions in MDA-MB-231 and MCF-7 breast cancer cells. After treatment of various concentrations of TCDD, cells were harvested with lysis buffer and western blot analyses were performed. The results showed an increase in PKC alpha expression and gap junctional protein, connexin 43, in the presence of TCDD in a concentration dependent manner. In translocation studies, PKC alpha was translocated from the cytoplasm to the membrane in the presence of 50nM or 100nM of TCDD. In addition, immunoprecipitation studies showed that 100 nM TCDD caused an increase in interaction of PKC alpha and connexin 43 compared to a control without TCDD treatment. We also found an increase in the activity of immunoprecipitated PKC alpha treated with 50 or 100 nM TCDD. These results suggest that TCDD may play a role in regulation of gap junctions in breast cancer cells.Understanding how TCDD affects gap junctional intercellular communication and subsequently interferes with cell communication of cancer cells will provide valuable evidence of the biochemical mechanisms of action. Future studies also include TCDD effect on other breast cancer cell lines like MDA-231, ZR-75 and T-47D, gap junctions and plaque formation using confocal microscopy. The effect of TCDD on the expression of other kinases will also be studied.

Abstract  The organic arsenical DMA-V is a bladder carcinogen in rats when administered in the diet or drinking water. Short-term administration of DMA-V causes urothelial cytotoxicity followed by regenerative hyperplasia. Long-term treatment of rats with the organic arsenical MMA-V in the drinking water caused urothelial hyperplasia but no bladder tumors. The inorganic arsenical arsenate was negative in a 2-year bioassay in rats. We evaluated the urothelial effects of treatment with 100 ug/g DMA-V or 100 ug/g MMA-V in the diet or 100 ug/g As-V in the drinking water. Rats were sacrificed after 1, 2, 6, 8, and 10 weeks of treatment and the effects on the rat bladder epithelium were determined by light and scanning electron microscopy (SEM). There was a decrease in body weight gain in the 100 ug/g As-V group during the first 2 weeks of treatment indicating that the dose of As-V used was toxic. However, this might have been related to an aversion to drinking the As-V-containing drinking water rather than actual toxicity. The mean achieved dosage of arsenic was higher in the As-V group (10.4 mg/kg) compared to the DMA-V (4.94 mg/kg) and MMA-V (4.88 mg/kg) groups partially due to a higher consumption of water per kg of body weight than diet. By light microscopy, there was no increase in urothelial hyperplasia in the DMA-V or MMA-V groups at any time point. However, there was an increase in urothelial hyperplasia in the As-V group as early as 1 week after the start of treatment. By SEM, there were significant changes in the bladder urothelium of rats treated with As-V or DMA-V. Only slight changes in the bladder urothelium were found in the group treated with MMA-V. Over time, the changes detected in the urothelium of the DMA-V-treated rats increased in severity but the changes in the urothelium of the As-V-treated rats did not. This is the first report of arsenate-induced changes in the rat bladder urothelium.

Abstract  Hexachlorobenzene (HCB), a widespread epigenetic environmental carcinogen, renders female more susceptible than male rats to development of hepatic tumors. We have previously shown that in female, but not male rats, HCB down-regulates gene expression and causes a significant decrease in gap junctional intercellular communication (GJIC). The latter results in part from a decrease in the liver gap junction protein, connexin 32 (Cx32). This study was aimed to assess the mechanism of HCB-induced Cx32 down-regulation by focusing on the binding of transcription factors the gene promoter. Male and female rats were administered 100 mg/kg of HCB by gavage for five consecutive days and killed 45 days later; control rats received corn oil alone. Liver nuclear proteins were isolated and incubated with labeled double stranded DNA from three regions of the Cx32 promoter (FR26, Fr53, Fr110) which have been shown to regulate the expression of hepatic Cx32. The binding protein-DNA complexes were analysed by Electrophoretic Mobility Shift Assay. No significant differences between groups were observed for the two complexes formed with Fr53; for Fr110 a significant 3-fold decrease in the intensity of the complex formed was observed between HCB-treated and control females, while no changes were observed in proteins isolated from male livers. Furthermore, a female-specific 5-fold decrease in binding was observed with Fr26. Supershift experiments are currently under investigation to confirm the identity of the transcription factors binding to each DNA fragment. Together, these data suggest that the decrease in Cx32 mRNA level induced by HCB in females results from a decrease in the binding of transcription factors which stimulate the expression of Cx32.

Abstract  Iconix Pharmaceuticals has developed a large-scale chemogenomics database, DrugMatrix, containing gene expression data from over 630 compounds administered to rats, along with their associated clinical pathology, histopathology and in vitro pharmacology screening data. Using gene expression data, we have mined a comprehensive library of Drug Signatures for the prediction of pathology, pharmacology and mechanism of action. To understand the molecular basis of these predictions, we have developed Motif technology. Using our SPLP classification algorithm, pathology, pharmacology and mechanism of action endpoints can be predicted on the basis of pathway information. By analogy, Drug Signatures are derived utilizing all informative genes on the microarray, while Motifs utilize only genes in a certain signaling pathway. In doing so, the unique state of multiple pathways can be now statistically linked to phenotypic endpoints, thus adding both classification and mechanistic understanding to predictions. Iconix has developed over 1000 validated Motifs in 5 different tissues, representing over 100 biological pathways. A study of Aflatoxin B1, a genotoxic hepatocarcinogen, illustrates the value of Motifs. Liver tissue from rats treated with 0.2 mg/kg/day of Aflatoxin B1 for 5 days was profiled, and the Drug Signatures and Motifs were applied to the gene expression data. Aflatoxin B1 treatment was predicted to match several Drug Signatures, including the Genotoxic Hepatocarcinogen signature. Motifs indicate that the hepatocarcinogenicity prediction can be explained by component pathways related to the DNA damage and apoptosis pathways, oxidative stress, IL-6 and p53 signaling. Unlike other pathway analysis tools, the state of a pathway can now be linked to different phenotypic endpoints, thereby providing both classification and mechanistic understanding to microarray expression data.

Abstract  Assessment of human liver damage after exposure to hepatotoxicants is best achieved by liver biopsy, a procedure accompanied by a significant amount of risk for the patient. Blood offers an accessible tissue which is significantly easier to sample for routine diagnoses. Serum enzymes that reflect serious liver damage are routinely used clinically, but no markers for incipient toxicity or exposure before manifestation of liver damage are available. We utilized the model hepatotoxicant acetaminophen (APAP) to test the hypothesis that blood can serve as a surrogate organ carrying gene expression signatures reflective of impending liver damage. We exposed Fisher 344 rats to sub-toxic (150mg/kg) and toxic (1500 and 2500 mg/kg) doses of APAP, sacrificed the animals 6, 12 or 24 hours after exposure and performed gene expression analysis on blood and liver specimens. Our analysis demonstrates that blood can carry signatures of incipient toxicity after sub-toxic and toxic exposure to APAP before the manifestation of liver damage measurable with clinical chemistry or histopathology. One example of such a signature contains genes involved in energy demanding pathways like gluconeogenesis and ATP-synthesis ?a potential indication of mitochondrial damage. Also, comparison of blood and liver gene expression changes resulted in both, gene expression alterations that are conserved between tissues (e.g. genes involved in interleukin-6 signaling) and changes unique to either tissue (e.g. genes involved in IGF-1 signaling were only in the liver differentially expressed, while those involved in Leukotriene metabolism only in blood). In summary, gene expression profiling revealed signatures in blood cells indicative of impending organ damage to the liver and provided new insights into common and diverse mechanisms of toxicity of APAP exposure in blood and liver.

Abstract  In previous work at the National Cancer Institute, Frederick, MD, the authors developed and produced an Internet-based tool that allows alignment of putative transcriptional regulatory regions for lists of genes such as those resulting from gene expression microarray experiments. Toxicological studies and similar experimental paradigms often yield lists of genes potentially co-regulated as based on their coordinate expression changes in response to the treatments. This poster describes the ABCC GRID Promoter TFSite Comparison Page. The authors used three examples, two based on research results from literature and one on an experiment they conducted for this study, to demonstrate that new hypotheses and research directions can be proposed by identification of potential transcription factor binding sites shared among a group of genes, the service the web page provides. Specific application of the webtool to toxicology in dose-response gene expression studies and similar treatment-response experimental paradigms is discussed, including limitations of the application. The views expressed in this poster are those of the authors and may not necessarily reflect the views or policies of the U. S. Environmental Protection Agency or the National Cancer Institute.

Abstract  Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) regulate detoxification and transport genes following chemical treatment and cholestatic liver disease. Mice deficient in these nuclear receptors exhibit changes in expression of target genes as well altered susceptibility to acetaminophen (APAP) hepatotoxicity. Toxic doses of APAP significantly induce hepatic expression of the mouse basolateral efflux transporter multidrug resistance protein 4 (Mrp4). The purpose of this study was to determine if CAR and PXR participate in the induction of Mrp4 following APAP administration. APAP (400 mg/kg i.p.) was administered to mice lacking CAR (CAR-/-) or PXR (PXR-/-), double knockouts (DKO), or wild-type (WT) mice. DKO mice were included in this study due to the documented redundancy of CAR and PXR receptors in the regulation of target gene expression. Hepatic gene and protein expression of Mrp4, and hepatotoxicity (serum ALT activity and histopathology) were assessed at 24 and 48 hrs. At 24 hrs after APAP, hepatotoxicity was greatest in the DKO mice compared to the WT and PXR-/- mice which had comparable liver injury. Hepatotoxicity was lowest in CAR-/- mice but the difference was not significant. By 48 hrs, no differences in liver injury were observed between the genotypes. APAP treatment increased Mrp4 mRNA in WT (5- fold), CAR-/- (6-fold), PXR-/- (3-fold), and DKO (4-fold) mice between 24 and 48 hrs. Western blot analysis demonstrated similar induction of Mrp4 protein (3 to 4-fold) in all genotypes 48 hrs after APAP. Up-regulation of Mrp4 occurred predominantly in centrilobular hepatocytes as determined by immunohistochemistry. In conclusion, alternative pathways independent of CAR and PXR are likely regulating the induction of Mrp4 during APAP liver injury.