The development of a real-time biosensor for the detection of trace levels of trinitrotoluene (TNT) in aquatic environments.

This paper describes the development of a highly sensitive TNT immunosensor consisting of a highly specific monoclonal antibody coupled with a prototype fluorescence-based detector system (KinExA Inline Biosensor, Sapidyne Instrument Inc). The antibody developed possesses a high affinity for TNT (association constant, aK 8.2) with minimal cross reactivity with other compounds such as tetryl, 2,4-dinitrotoluene and 2-amino-4,6-dinitrotoluene. This system provides sample assessment within 160 s from source acquisition and possesses sensitivity for TNT of 0.05 microg/L in ground water. The sensor can be regenerated in 8 min, allows a minimum of 40 repeated readings, and has a standard error of 0.1-0.4% between repeat readings. The fluidics and software allow samples to be obtained from up to eight different sources allowing the user to examine the stratification of the pollutant in the water column. We believe that this immunosensor can be used to rapidly assess trace levels of TNT in environmental water samples.

Characterization of adjacent breast tumors using oligonucleotide microarrays.

BACKGROUND: Current methodology often cannot distinguish second primary breast cancers from multifocal disease, a potentially important distinction for clinical management. In the present study we evaluated the use of oligonucleotide-based microarray analysis in determining the clonality of tumors by comparing gene expression profiles. METHOD: Total RNA was extracted from two tumors with no apparent physical connection that were located in the right breast of an 87-year-old woman diagnosed with invasive ductal carcinoma (IDC). The RNA was hybridized to the Affymetrix Human Genome U95A Gene Chip (12,500 known human genes) and analyzed using the Gene Chip Analysis Suite 3.3 (Affymetrix, Inc, Santa Clara, CA, USA) and JMPIN 3.2.6 (SAS Institute, Inc, Cary, NC, USA). Gene expression profiles of tumors from five additional patients were compared in order to evaluate the heterogeneity in gene expression between tumors with similar clinical characteristics. RESULTS: The adjacent breast tumors had a pairwise correlation coefficient of 0.987, and were essentially indistinguishable by microarray analysis. Analysis of gene expression profiles from different individuals, however, generated a pairwise correlation coefficient of 0.710. CONCLUSION: Transcriptional profiling may be a useful diagnostic tool for determining tumor clonality and heterogeneity, and may ultimately impact on therapeutic decision making.

Recent advances in breast cancer biology.

Approximately 1 in 10 US women will be diagnosed with breast cancer in her lifetime. With such a high incidence, breast cancer is a serious health concern for all American women. Within the past year, clues about the function of genes associated with breast cancer have been garnered, and novel genes that may contribute to breast tumorigenesis have been discovered. In addition, unique animal models and improvements in gene expression profiling technology have given researchers new tools to address previously unanswerable questions about this disease.

Screening for genomic rearrangements in families with breast and ovarian cancer identifies BRCA1 mutations previously missed by conformation-sensitive gel electrophoresis or sequencing.

The frequency of genomic rearrangements in BRCA1 was assessed in 42 American families with breast and ovarian cancer who were seeking genetic testing and who were subsequently found to be negative for BRCA1 and BRCA2 coding-region mutations. An affected individual from each family was tested by PCR for the exon 13 duplication (Puget et al. 1999a) and by Southern blot analysis for novel genomic rearrangements. The exon 13 duplication was detected in one family, and four families had other genomic rearrangements. A total of 5 (11. 9%) of the 42 families with breast/ovarian cancer who did not have BRCA1 and BRCA2 coding-region mutations had mutations in BRCA1 that were missed by conformation-sensitive gel electrophoresis or sequencing. Four of five families with BRCA1 genomic rearrangements included at least one individual with both breast and ovarian cancer; therefore, 4 (30.8%) of 13 families with a case of multiple primary breast and ovarian cancer had a genomic rearrangement in BRCA1. Families with genomic rearrangements had prior probabilities of having a BRCA1 mutation, ranging from 33% to 97% (mean 70%) (Couch et al. 1997). In contrast, in families without rearrangements, prior probabilities of having a BRCA1 mutation ranged from 7% to 92% (mean 37%). Thus, the prior probability of detecting a BRCA1 mutation may be a useful predictor when considering the use of Southern blot analysis for families with breast/ovarian cancer who do not have detectable coding-region mutations.

Monolithic microfabricated valves and pumps by multilayer soft lithography.

Soft lithography is an alternative to silicon-based micromachining that uses replica molding of nontraditional elastomeric materials to fabricate stamps and microfluidic channels. We describe here an extension to the soft lithography paradigm, multilayer soft lithography, with which devices consisting of multiple layers may be fabricated from soft materials. We used this technique to build active microfluidic systems containing on-off valves, switching valves, and pumps entirely out of elastomer. The softness of these materials allows the device areas to be reduced by more than two orders of magnitude compared with silicon-based devices. The other advantages of soft lithography, such as rapid prototyping, ease of fabrication, and biocompatibility, are retained.

Effect of in vitro exposure to tributyltin on generation of oxygen metabolites by oyster hemocytes.

Mollusks depend chiefly on hemocyte-mediated cytotoxic mechanisms such as reactive oxygen species (ROS) to defend against pathogenic microorganisms. The effect of in vitro tributyltin chloride (TBT) exposure on ROS generation by oyster (Crassostrea virginica) blood phagocytes is quantified in this study. Luminol-augmented chemiluminescence (LCL) was used to measure ROS activity of resting and zymosan-stimulated cells after 1 or 20 hr TBT exposure. LCL is thought to measure primarily the activity of the myeloperoxidase/hydrogen peroxide/ halide antimicrobial pathway. Hemocytes in TBT-free medium (controls) produced low level LCL, which was markedly stimulated by the addition of zymosan particles. Both resting and zymosan-stimulated LCL values were significantly inhibited by > or = 80 ppb TBT after either 1 or 20 hr of exposure. Exposure to < or = 2 ppb TBT concentrations for 20 hr produced slightly enhanced LCL activity, suggesting a hormesis-like effect. Partial reversibility of TBT suppression of LCL took place when previously exposed cells were put in TBT-free medium. The TBT concentrations used in these studies were not cytolethal in vitro and were considerably less than oyster tissue levels recorded after chronic, sublethal in vitro exposures. The data suggest that the common aquatic contaminant TBT can interact rapidly with C. virginica hemocytes to produce a partially reversible immunotoxicological lesion. Xenobiotic-induced suppression of ROS production by hemocytes may increase host susceptibility to infectious diseases.

Butyltin and copper monitoring in a Northern Chesapeake Bay marina and river system in 1989: An assessment of tributyltin legislation.

This study was designed to: (1) evaluate dibutyltin (DBT) and tributyltin (TBT) bi-weekly in the water column for four months during the peak boating season (June-September, 1989) at seven stations in the Back Creek and Severn River area of Maryland waters of Chesapeake Bay; (2) compare butyltin values from the 1989 study with values obtained from a similar butyltin monitoring study conducted in 1988 (after Maryland TBT legislation) and 1986 (before Maryland TBT legislation); (3) determine the extent of TBT paint use in the Back Creek area by surveying boat owners; (4) determine dissolved copper concentrations from three of the seven stations bi-weekly during the four-month study; and (5) compare dissolved copper concentrations at these stations with previous copper data collected in 1988.Mean four-month DBT concentrations ranged from 10 to 73 ng/L at the seven stations. Highest DBT concentrations occurred at Station 1 in a marina; lowest concentrations occurred at Station 7 in the Severn River. Mean four-month TBT concentrations ranged from 177 ng/L at Station 1 (marina) to 21 ng/L at Station 7 (Severn River). Maximum TBT concentrations of 361 and 570 ng/L occurred at marina SDtations 1 and 3, respectively. Temporal trends in both DBT and TBT (station mean concentrations by date) showed that peak concentrations occurred during the early part of the boating season followed by reductions in late summer and early fall. Spike concentrations of both DBT (117 and 62 ng/L) and TBT (308 and 366 ng/L) were reported on two sampling dates near a boat maintenance facility in Back Creek.There was a significant reduction in DBT concentrations from 1986 to 1989 when date was treated as a fixed effect. However, TBT concentrations were not significantly reduced between 1986 and 1989 when mean concentrations of TBT were averaged across stations and dates for each year. A significant reduction was reported at Station 1 (marina station) when each station was examined for differences between years. TBT was also reported to significantly decrease (p=0.0442) at Station 7 between 1988 and 1989. A boat owner survey in the study area showed that 6% of the recreational boats that were surveyed were painted with TBT paint in 1989. This was a significant decrease in TBT paint use from the previous year when 31% of recreational boat owners surveyed used TBT paints.An evaluation of dissolved copper concentrations at three stations in the study area in 1989 showed that mean concentrations from bi-weekly sampling for four months was 10 µg/L at Station 1, 7.8 µg/L at Station 4 and 2.7 µg/L at Station 7. Copper concentrations decreased with distance away from the Back Creek marinas. Copper concentrations at all three stations were significantly lower in 1989 than in 1988.

A selective ion monitoring assay for tributyltin and its degradation products.

Tributyltin (TBT) is a biocide which has been shown to enter the aquatic environment by release from antifouling paints. TBT is acutely toxic to some marine organisms at concentrations near 1 microgram l-1 and physiological changes may occur at low nanogram per liter concentrations. Gas chromatography/mass spectrometry (GC/MS) (methane chemical ionization) has been used for identification (full scanning) and quantification (selected ion monitoring) of TBT, dibutyltin (DBT) and monobutyltin (MBT). The butyltins were extracted from environmental water samples with hexane/0.2% tropolone and derivatized with hexyl magnesium bromide to form hexylbutyltins. Selected ion monitoring was at m/z 319 (TBT) and m/z 347 (DBT, MBT and tripentyltin, the internal standard). Calibration curves prepared in natural water were linear and detection limits were less than 2 ng l-1. GC/MS and GC with flame photometric detection were compared as quantification methods for environmental samples and were shown to give similar results at the low nanogram per liter levels.