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.
Fredrickson
H L
HL
US Army Engineer Research and Development Center, Environmental Laboratory, Waterways Experiment Station, Vicksburg, MS 39180-6199, USA. fredrih@wes.army.mil
Perkins
E J
EJ
Bridges
T S
TS
Tonucci
R J
RJ
Fleming
J K
JK
Nagel
A
A
Diedrich
K
K
Mendez-Tenorio
A
A
Doktycz
M J
MJ
Beattie
K L
KL
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000187:drug effects / Q000235:genetics*; D017931:DNA Primers; D004782:Environmental Health; D004784:Environmental Monitoring / Q000295:instrumentation / Q000379:methods*; D004798:Enzymes / Q000235:genetics; D004867:Equipment Design; D004926:Escherichia coli / Q000235:genetics; D023281:Genomics / Q000295:instrumentation / Q000379:methods*; D015386:Hazardous Substances / Q000032:analysis* / Q000633:toxicity; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D015345:Oligonucleotide Probes; D011077:Polychaeta / Q000235:genetics; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D013816:Thermodynamics; D014116:Toxicology / Q000295:instrumentation / Q000379:methods*
D017931:DNA Primers; D004798:Enzymes; D015386:Hazardous Substances; D015345:Oligonucleotide Probes; D011506:Proteins; D012333:RNA, Messenger; D004247:DNA