An Enlightened Approach to Screening for Dioxins

[Jackie Hunt Christensen <jchristensen@igc.apc.org>]

  >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
  cells luminesce.
  
                         [graphic]       
  
       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
       samples.
  
  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)
  612-870-4846 (fax)
  e-mail: <jchristensen@iatp.org>
  IATP's Endocrine Disrupter Resource Center: http://www.sustain.org/edrc