Shortcomings in USEPA's Approach for Predicting Risk Due to Consumption of Animal Food Products Impacted by Air Emissions from Hazardous Waste Combustion Facilities: A Case Study Involving Phthalates

As a part of the permitting process for hazardous waste combustion facilities, regulatory agencies are now conducting site-specific, multipathway risk assessments. In accordance with the approach established by the USEPA, the Texas Natural Resource Conservation Commission uses a prospective risk assessment paradigm whereby site-specific activity pattern and land use information is used to determine plausible exposure scenarios and pathways. A set of exposure scenarios defined as receptors (i.e., resident adult, resident child, farmer adult, farmer child, fisher adult and fisher child) is then assumed to be exposed via multiple applicable exposure pathways. In conducting such risk assessments, modeled air emissions of di-n-octyl phthalate (DNOP), at concentrations near or below detectable levels, have been observed to produce an unacceptable hazard in the farmer exposure scenario. Sensitivity analyses indicated that two key parameters affected hazard estimates for DNOP in the farmer scenario: the octanol-water partition coefficient (K_ow), which is used to predict bioaccumulation in animal tissue, and the metabolism factor, which is used to account for metabolism and elimination. Evidence indicates that K_ow values for highly lipophillie compounds are accurately determined using a slow-stir method. In addition, evidence indicates that the phthalates of interest are extensively metabolized and eliminated. However, current USEPA guidance includes geometric mean K_ow values for highly lipophillie compounds derived in part on methods that are outdated and no longer considered accurate. In addition, USEPA guidance only considers metabolism for bis-% ethylhexyl phthalate (BEHP). Collectively, these two shortcomings in the USEPA approach result in a 38-fold underestimation of hazard for BEHP and a 172,000-fold overestimation of hazard for DNOP.     

Monitoring for Heterosigma akashiwo using a sandwich hybridization assay

Field testing of a ribosomal RNA (rRNA)-targeted sandwich hybridization assay (SHA) for Heterosigma akashiwo (Raphidophyceae) in Puget Sound, WA, USA, has showed that the lower limit of detection is well below the level at which cells pose a danger to fish. Moreover, the assay has proven to be both rapid and easy-to-use. Isolates of H. akashiwo from Australia, Japan, New Zealand, South Korea, Spain and USA were correctly identified using the SHA, indicating that this diagnostic tool could be deployed globally. Samples containing H. akashiwo can be preserved for subsequent SHA analysis using several methods: fixation with acidic Lugol’s iodine followed by room temperature storage, collection onto Durapore filters followed by storage at −70 °C or, alternatively, the filters are mixed with a lysis solution buffer and the sample lysate stored at −70 °C. Additionally, we sought to determine whether the SHA could successfully detect H. akashiwo in the presence of clay that might some day be used to mitigate the impacts of natural H. akashiwo blooms. Results from preliminary laboratory trials indicate that clay at the maximum proposed dosage rate does not interfere with the assay. Thus, it may be possible to use the SHA as a simple means of following the fate of H. akashiwo cells during larger-scale clay mitigation trials.     

As(III) inhibits ultraviolet radiation-induced cyclobutane pyrimidine dimer repair via generation of nitric oxide in human keratinocytes

Inorganic arsenic enhances skin tumor formation when combined with other carcinogens including ultraviolet radiation (UVR). The inhibition of DNA damage repair by arsenic has been hypothesized to contribute to the cocarcinogenic activities of arsenic observed in vivo. Cyclobutane pyrimidine dimers (CPDs) are an important mutagenic UVR photoproduct and implicated in the genesis of nonmelanoma skin cancer. The current study demonstrates that low concentrations of arsenite (As(III)) inhibit UVR-induced CPD repair in a human keratinocyte cell line via nitric oxide (NO) and inducible nitric oxide synthase (iNOS). Following As(III) treatment, NO production and iNOS expression are elevated. Little is known about regulation of iNOS by As(III) and further investigations indicated that p38 mitogen-activated protein kinase (p38 MAPK) and NF-kappaB are required for As(III) induction of iNOS expression. This As(III)-stimulated signaling cascade was involved in inhibition of UVR-induced CPD repair as disruption of p38 MAPK activity and NF-kappaB nuclear translocation counteracted the effects of As(III) on CPD repair. Selective inhibition of iNOS ameliorated As(III) inhibition of CPD repair, thereby suggesting that iNOS is a downstream mediator of As(III) activity. These findings provide evidence that an As(III)-stimulated signal transduction cascade culminating in elevated iNOS expression and NO generation is an underlying mechanism for inhibition of UVR-induced DNA damage repair by arsenic.     

Dexamethasone and GLP-2 administered to rat dams during pregnancy and lactation have late effects on intestinal sugar transport in their postweaning offspring

Intestinal function in young animals is influenced by maternal factors, such as alterations in the maternal diet. Glucagon-like peptide 2 (GLP-2) enhances intestinal growth and absorption in mature animals. Glucocorticosteroids induce intestinal maturation in neonates and increase sugar uptake in adult animals. It is not known if maternally administered GLP-2 or glucocorticosteroids have persistent effects on intestinal transport in the offspring. This study was undertaken to determine (1) the influence of maternal GLP-2, dexamethasone (DEX) and GLP-2+DEX on intestinal sugar uptake in postweaning offspring and (2) if alterations in uptake are due to variations in intestinal morphology, sugar transporter abundance or the abundance of selected signals. Nursing rat dams were treated during pregnancy and lactation with GLP-2 (0.1 mug/g per day sc), DEX (0.128 microg/g per day sc), GLP-2+DEX or placebo. The offspring were sacrificed 4 weeks after weaning, and glucose and fructose uptake was determined using an in vitro intestinal ring uptake technique. sodium-dependent glucose transporter, glucose transporter (GLUT) 5, GLUT2, sodium potassium adenosine triphosphatase and selected signals were assessed by immunohistochemistry. The treatments did not affect body weights or intestinal morphology. GLP-2 and GLP-2+DEX increased jejunal fructose uptake, and GLP-2+DEX increased the jejunal and ileal maximal transport rate for glucose uptake. Protein kinase B and mammalian target of rapamycin abundance were also increased, while transporter abundance was unchanged. We speculate that these alterations in sugar uptake may be due to changes in the intrinsic activity of the transporters mediated by the phosphatidylinositol-3-kinase pathway. These alterations in uptake may have nutritional implications for the offspring of mothers who may be treated with GLP-2 or glucocorticosteroids.     

Discovery of potent, orally active benzimidazole glucagon receptor antagonists

The discovery and optimization of potent and selective aminobenzimidazole glucagon receptor antagonists are described. One compound possessing moderate pharmacokinetic properties in multiple preclinical species was orally efficacious at inhibiting glucagon-mediated glucose excursion in transgenic mice expressing the human glucagon receptor, and in rhesus monkeys. The compound also significantly lowered glucose levels in a murine model of diabetes.     

Integrating microbiological, microsensor, molecular, and physiologic techniques in the study of coral disease pathogenesis

The study of coral diseases requires an integrated approach that includes a combination of field and laboratory methods. By combining and building upon information available from multiple disciplines, within both field and laboratory applications, we have been successful in characterizing a number of coral diseases. To illustrate the utility of the integrative approach two very different coral diseases, black band disease and plague, are discussed in detail. Comparison of our ongoing characterization of each disease demonstrates that, within the integrative approach, different combinations of microbiological, microsensor, molecular, and physiologic techniques are required. The pathobiology of black band disease, which consists of a complicated, synergistic microbial consortium functioning around a dynamic sulfur cycle, is slowly being unraveled using a combination of methods. Our study of plague, on the other hand, has progressed in a very different manner that is controlled by the fact that this disease has, to date, emerged in three forms on reefs of the Florida Keys. The study of plague types I, II, and III will be detailed to illustrate the difficulty of characterizing a disease that rapidly evolves in the natural environment of the reef. Our ongoing study of additional (also very different) coral diseases will be summarized from the perspective of combined methodologies to illustrate the range and magnitude of questions that must be addressed and answered in order to understand coral disease pathogenesis and thus coral disease etiology.     

Accelerated production and identification of fertile,homozygous transgenic wheat lines by anther culture

Anther culture has been developed in the winter wheat cultivar Florida to achieve accelerated production and identification of homozygous transgenic lines. With untransformed, seed-derived plants to develop the culture system, it was shown that cold pre-treatment of spikes excised from donor plants and addition of 2,4-dichlorophenoxyacetic acid together with either kinetin or 6-benzylaminopurine in the callus induction medium improves the anther culture response. The procedure developed allowed production of fertile homozygous lines within 8–9 months, which includes an 8-week vernalisation period. With transgenic wheat plants produced by particle bombardment as donors, we show that the system can be used to produce homozygous transgenics, requiring one generation cycle. Both T₀ tissue culture-derived plants and their T₁ seed-derived descendents serve as suitable donors. We show that an anther culture response comparable to that of untransformed, seed-derived plants can be achieved with T₀ tissue culture-derived plants. PCR and Southern molecular analyses of anther culture-derived transgenics show that the transgenes are stably inherited; there are no perturbations at the chromosomal level around the sites of transgene integration as a result of in vitro chromosome manipulation during anther culture.