Superfund Research Program
Optimization of the CALUX Bioassay for Use in Detecting Dioxin and Related Chemicals in Serum
Polycyclic and halogenated aromatic hydrocarbons (PAHs/HAHs) are important classes of environmental contaminants commonly found at hazardous waste sites. They are known to cause a variety of adverse health effects in humans and wildlife. Most current methods for detection of these chemicals are rather expensive and slow. Researchers at the University of California-Davis have been exploring the utility of bioassay systems for the direct detection of PAHs/HAHs in blood samples. These studies resulted in the development and optimization of a cell bioassay system that is both quick and inexpensive. This recombinant cell bioassay has been engineered to respond to dioxin-like HAHs and PAHs, with the activation of gene expression, specifically that of firefly luciferase. This chemically-activated luciferase expression (CALUX) assay responds to these chemicals in a dose-, time-, and chemical-specific manner, and as such, the assay is a reliable detector of luciferase activity resulting from exposure to dioxin-like chemicals.
The investigators developed the CALUX bioassay and have optimized its performance to allow for rapid and direct analysis of small volumes (25-50 ul) of whole serum. The assay utilizes a 96-well microtiter plate and does not require solvent extraction of the sample. The optimal volume of serum needed for analysis was determined through tests with sterile goat serum containing dioxins and other chemicals. Preparation and analysis time--from initial cell treatment to final analysis--has been minimized, and an assay can be completed in approximately five hours. The optimized assay includes simple procedures to correct for interplate and intraplate variability, and allows for direct comparison of results within and among plates with a great degree of confidence. In validation studies, significant dose-dependent results for eight chemicals (TCDD, TCDF, PeCDF, TCB, 3-PeCB, BKF, BAP and BAA) were observed. Previous studies have documented the utility and validity of the CALUX bioassay to detect these chemicals in a variety of environmental and biological matrices.
Based on the parameters of the assay, CALUX can be used as a screening tool for measurement of HAHs/PAHs and related chemicals in small volumes of whole serum. As an example application of this bioassay system, a subset of sera from several different marine wildlife species (with unknown xenobiotic exposure) was analyzed using the CALUX bioassay. Results show a spectrum of induced luciferase activity that can be interpreted based roughly on the natural histories of each. Samples collected from black-footed albatross, a seabird that primarily resides at a distance from shore-based activities, had minimal luciferase activity. Double-crested cormorants, a species that has more interaction with the mainland (therefore more potential contact with relevant xenobiotics) exhibited statistically higher levels of induction but at relatively low levels (on the order of picomolar equivalents of TCDD). The final species examined, the Southern sea otter, is a species at high risk of pollutant exposure, and the data indicate that the otters have been exposed to relatively high levels of unknown pollutants (at a concentration approximately equivalent to that of 0.1 nM TCDD).
CALUX can be used as a highly effective method for screening large numbers of potentially exposed individuals to broad classes of chemicals such as PAHs, HAHs and endocrine dusruptors and can be performed rapidly and inexpensively. Positive samples can be chemically analyzed to determine the specific chemicals and congeners present when warranted. Subsequent studies will focus on final validation of this bioassay approach by comparing the CALUX induction results with those of analytical chemical analysis (GC/MS) of the same unknown sample.
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To learn more about this research, please refer to the following sources:
- Ziccardi MH, Gardner IA, Denison MS. 2000. Development and modification of a recombinant cell bioassay to directly detect halogenated and polycyclic aromatic hydrocarbons in serum. Toxicol Sci 54(1):183-193. PMID:10746945
- Murk AJ, Leonards PE, Bulder AS, Jonas AS, Rozemeijer MJ, Denison MS, Koeman JH, Brouwer A. 1997. The CALUX (chemical-activated luciferase expression) assay adapted and validated for measuring TCDD equivalents in blood plasma. Environ Toxicol Chem 16(8):1583-1589.
- Murk AJ, Legler J, Denison MS, Giesy JP, van der Guchte CJ, Brouwer A. 1996. Chemical-activated luciferase gene expression (CALUX): a novel in vitro bioassay for Ah receptor active compounds in sediments and pore water. Fundam Appl Toxicol 33(1):149-160. PMID:8812260
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