Towards environmental toxicogenomics -- development of a flow-through, high-density DNA hybridization array and its application to ecotoxicity assessment.

Assessment of the environmental hazard posed by soils/sediments containing low to moderate levels of contaminants using standard analytical chemical methods is uncertain due (in part) to a lack of information on contaminant bioavailability, the unknown interactive effects of contaminant mixtures, our inability to determine the species of a metal in an environmental matrix, and the relative sensitivity of bioassay species. Regulatory agencies compensate for this uncertainty by lowering cleanup goals, but in this process they effectively exclude otherwise attractive cleanup options (i.e. bioremediation). Direct evaluations of soil and sediment toxicity preclude uncertainty from most of these sources. However, the time and cost of chronic toxicity tests limits their general application to higher levels of tiered toxicity assessments. Transcriptional level (mRNA) toxicity assessments offer great advantages in terms of speed, cost and sample throughput. These advantages are currently offset by questions about the environmental relevance of molecular level responses. To this end a flow-through, high-density DNA hybridization array (genosensor) system specifically designed for environmental risk assessment was developed. The genosensor is based on highly regular microchannel glass wafers to which gene probes are covalently bound at discrete (200-microm diameter spot) and addressable (250-microm spot pitch) locations. The flow-through design enables hybridization and washing times to be reduced from approximately 18 h to 20 min. The genosensor was configured so that DNA from 28 environmental samples can be simultaneously hybridized with up to 64 different gene probes. The standard microscopic slide format facilitates data capture with most automated array readers and, thus high sample throughput (> 350 sample/h). In conclusion, hardware development for molecular analysis is enabling very tractable means for analyzing RNA and DNA. These developments have underscored the need for further developmental work in probe design software, and the need to relate transcriptional level data to whole-organism toxicity indicators.

Definitive characterization of uric acid as an interferent in peroxidase indicator reactions and a proposed mechanism of action.

We have characterized and identified uric acid as an interferent to peroxidase catalyzed reactions where hydrogen peroxide is generated at relatively low concentrations. The implications of these findings are important for those utilizing peroxidase as an indicator reaction where low primary substrate concentrations require their preliminary extraction or chemical modification. We have shown that the elimination of uric acid as an interferent from biologic fluid obviates the necessity for such treatment. In amniotic fluid, our data suggests that uric acid represents the only interference to peroxidase-catalyzed reactions, especially when using p-substituted phenols as proton donors. The removal of uric acid has been shown to eliminate hydrogen peroxide reduction and should allow for an increase in sensitivity and specificity for measurements incorporating a peroxidase-coupled indicator reaction, hence, more effective use of these reaction sequences. To our knowledge, this is the first report of a mechanism to eliminate hydrogen peroxide reduction in amniotic fluid.

Assessment of phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin in human serum.

We describe a method for analyzing the choline-containing phospholipids, namely phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin that is highly sensitive, easy to perform, and very reliable. The method employs enzymic hydrolysis of choline from these phospholipids by phospholipase D with subsequent oxidation of choline by choline oxidase and generation of hydrogen peroxide. Hydrogen peroxide is then reduced by peroxidase with p-hydroxy-phenylacetic acid acting as an oxygen acceptor to form a fluorescent product which is stable for at least 24 h without loss of linearity. The analysis requires a 5 microL sample volume and demonstrates linearity over a wide range (133 mumol/L-6.65 mmol/L). The CVs are less than 3%.