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An Enlightened Approach to Screening for Dioxins
>From NOvember ENVIRONMENTAL HEALTH PERSPECTIVES "Forum"
"An Enlightened Approach to Screening for Dioxins
Scientists recently had a bright idea about how to screen for environmental
toxins. Researchers at the University of California at Davis have developed
a bioassay system to detect polyhalogenated aromatic hydrocarbons such as
dioxins in environmental samples. Dubbed the CALUX (for chemically
activated luciferase gene expression) system, the assay is based on
recombinant cell lines into which researchers have inserted the firefly
luciferase gene. When exposed to dioxin-like compounds, the recombinant
The glow gives it away. A new assay uses the firefly luciferase
reporter gene, which luminesces in the presence of the Ah
receptor, to test for the presence of dioxins in environmental
Polyhalogenated aromatic hydrocarbons are a diverse group of compounds that
are widespread in the environment. Exposure to these compounds can lead to
carcinogenesis, liver toxicity, birth defects, damage to the immune system,
skin lesions, and even death. "Given the ubiquitous presence of these toxic
compounds in the environment, there is a need for a rapid, inexpensive
screening assay to monitor toxic output at a given site, to detect the
presence of these chemicals in individuals who work in such environments,
and to test sites in which these chemicals are believed to be deposited,"
says Michael Denison, professor of environmental toxicology at the
University of California at Davis and one of the assay's inventors.
Denison and his colleagues have studied human, rat, guinea pig, hamster,
and mouse cells, and are currently experimenting with a fish cell line.
Testing is carried out by placing the environmental specimen in a test
plate with the recombinant cells. The cells contain the luciferase reporter
gene, which is linked to a DNA sequence called a dioxin-responsive element
(DRE). The DRE is the binding site for the dioxin-activated aryl
hydrocarbon receptor (AhR), a cell protein that mediates the toxic effects
of dioxins. When the cells are exposed to dioxin-like compounds present in
environmental samples, the AhR is activated and stimulates expression of
the luciferase gene via the DRE. Luciferase can be easily measured because
it emits light.
EPA regulations require an assay of the concentration of individual
dioxin-like compounds in an environmental sample using high-resolution gas
chromatography coupled with high-resolution mass spectrometry. Results for
the individual compounds are then multiplied by a toxic equivalency factor
to arrive at the total toxic equivalency for the mixture of toxins. This is
a slow, cumbersome, and expensive process. The CALUX system detects the
presence of such chemicals, but does not indicate which individual chemical
or combination is present. Still, it is a rapid and inexpensive screening
method, Denison explains.
"This [system] is a natural progression from the current bioassay," says
Denison. "The previous bioassay, [using] the H4IIE wild-type cells, lacks
much of the selectivity and sensitivity that the CALUX system has. Our
system has less 'background noise' than does the H4IIE method, which
measures activity from many compounds not [chemically] related to dioxin."
The bioassay is so promising that it is now the object of a commercial
enterprise. George Clark, president of Xenobiotic Detection Systems, Inc.
in Durham, North Carolina, is marketing the assay to environmental
researchers. "Currently we're offering analyses of blood, serum, milk,
water, or sediment in parts per trillion of
2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents. We're seeking regulatory
approval for our assay as a screening method that can be confirmed by gas
chromatographymass spectrometry, which should provide significant savings
in the analysis of the toxicity of this class of compounds," Clark says.
The bioassay is not a test that can currently be run in the field, but
specimens can be collected, frozen, and forwarded to the laboratory for
assay. "However," says Clark, "we're developing a mobile laboratory that
can be parked at the site of investigation in areas that require a large
number of tests."
"The strong point of this assay is its speed and potential as a screening
method," according to Scott Masten, a fellow in the Environmental
Toxicology Program at the NIEHS who is familiar with the assay. "Using the
chromatographspectrometer method you could assay perhaps two dozen
specimens in a week. This bioassay can screen a hundred specimens in a week
and only those that show activity, that luminesce, need to be run through
the chromatograph and spectrometer. The bioassay will be even faster if it
is automated, which can be done. The major disadvantage of the CALUX method
is the problem with specificity. It measures total dioxin-like activity in
the specimen without indicating which chemicals are present. The active
sample still must be analyzed by chromatograph and spectrometer, but even
so, the bioassay is around 20% the cost of the chromatographspectrometer."
Jackie Hunt Christensen
Food Safety Project Director
Institute for Agriculture and Trade Policy
2105 1st Avenue South
Minneapolis, MN 55404
612-870-3424 (direct line)
IATP's Endocrine Disrupter Resource Center: http://www.sustain.org/edrc