8070

8070 P450 REPORTER GENE RESPONSE TO DIOXIN-LIKE ORGANIC COMPOUNDS*
8070 A. Introduction
1. General Discussion
A cell culture of human liver cancer cells can be used to detect
the presence of toxic and/or carcinogenic organic compounds in
environmental samples. A sample of water, soil, aquatic sedi-
ment, or tissue is extracted with a solvent to remove semivolatile
organic compounds, and a small amount of the extract is applied
to a culture well containing cells attached to the bottom of the
well in medium. After exposure, the cells are rinsed and lysed,
the cell fragments are removed via centrifugation, and the extract
is tested for luminescence. The cells produce a luminescent
enzyme (luciferase) if the extract contained dioxin-like com-
pounds because a reporter gene (plasmid) from the firefly has
been attached to the human chromosome at the site induced by
dioxin and other planar compounds (at the CYPlAl gene). The
amount of light produced, which is quantified by a luminometer,
is a function of the concentrations and induction potency of the
organic compounds in the extract. Dioxin [2,3,7,8-tetrachloro-
dibenzo-p-dioxin (TCDD)] has the strongest affinity for the
receptor on the cell membrane (Ah-receptor), and therefore will
be detected at the lowest concentration.
This assay is both a detection system and a meaningful bio-
logical response to the toxicants in environmental samples. It can
be used to screen environmental samples for some of the most
toxic and carcinogenic compounds. Only compounds that are
dioxin-like and attach to the Ah-receptor will induce the CYPlAl
gene and result in the production of luciferase. Such induction
would occur in humans or wildlife, including aquatic species, if
these compounds came in contact with their tissues. Inducing the
CYPlAl gene is one of the key factors used in designating a
compound a carcinogen. High levels of such induction in fish
(P450 measurements) have been shown to correlate with histo-
logical damage and reduced reproductive capacity.
Another procedure based on these principles is available.
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2. Reference
1. AMERICAN SOCIETY FOR TESTING AND MATERIALS. 2009. Guide for
measuring the presence of planar organic compounds which induce
CYP1A, using reporter gene test systems, E1853 M-98. Annual
Book of ASTM Standards, Vol. 11.05. American Soc. Testing &
Materials, W. Conshohocken, Pa.
8070 B. The P450 RGS Test
1. Principle and Significance
The P450 Reporter Gene System (RGS) is a biomarker test for
detecting toxic and/or carcinogenic organic compounds, using a
transgenic cell line (101L)* derived from the human hepatoma
cell line (HepG2). Under appropriate test conditions, induction
of the CYPlAl gene in mammalian cells normally results in the
production of the enzyme P450lAl. This response is evidence
that the cells have been exposed to one or more xenobiotic
organic compounds, including dioxins, furans, coplanar poly-
chlorinated biphenyls (PCBs), and several polycyclic aromatic
hydrocarbons (PAHs). Detection of induction has been made
rapid and inexpensive by the stable integration of a firefly
plasmid, such that Ah-receptor binding and subsequent transcrip-
tion results in the production of luciferase. This RGS test has
shown concentration–response relationships using dilutions of
TCDD, 2,3,7,8-TCDF, five coplanar PCBs, and eight PAHs, and
has responded to application of extracts from environmental
samples.
1– 4
Environmental samples (water, tissue, soil, or
aquatic sediments) may be extracted via established methods,
and the extracts applied to the test system to detect toxic and
carcinogenic organic compounds.
The organic compounds that induce the CYPlAl site on the
chromosome are toxic, often carcinogenic, and several have been
shown to bioconcentrate and biomagnify. Various birds, mam-
mals, and fish exposed to these compounds have exhibited phys-
iological, reproductive, and histopathological effects.
6–7
Mil-
lions of dollars are spent annually on chemical analyses of water,
food, wildlife, soil, and aquatic sediment because of concern for
contamination from these compounds. Using a screening tool,
such as RGS, will permit selection of a subset for further
chemical characterization.
2. Test Summary
Details of the culture and testing methods have been pub-
lished.
8 –10
For most environmental samples, dichloromethane
(DCM) is used as the extraction solvent, but for extraction of ash
samples for dioxins/furans, toluene is used. Initial dry weight
(determined on a separate subsample) of extracted sample, final
volume of solvent containing the extracted material (0.5 or 1.0
mL), and amount applied to the cells are recorded. Regardless of
the extraction solvent or system, DMSO, iso-octane, or another
* Approved by Standard Methods Committee, 2001. Editorial revisions, 2010.
Joint Task Group: Donald J. Reish (chair).
* Cells are available from Columbia Analytical Services, Vista, CA 92083.
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low-toxicity solvent is the final solvent applied to the cells.
Extracts of environmental samples are added to individual wells
(six-well plates) containing approximately 1 million cells that
have been growing for 3 d. Usual exposure time is 16 h. To
examine the relationship between chlorinated hydrocarbons and
PAHs in an extract, measure response at 6 and 16 h, because
PAHs reach peak induction at 6 h and PCBs and dioxins reach
peak induction at 16 h.
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If information is required on only the
chlorinated inducers (dioxins, furans, coplaner PCBs) first clean
the extracts of PAHs with silica gel columns. Application of 20
␮ L solvent produces a low background (control) induction when
applied to cells in 2 mL of culture medium. The luminescence [in
relative light units (RLU)] of the combined cytoplasm from the
cells in each well is compared to that of other replicate wells, the
solvent control, and reference toxicants (TCDD and five concen-
trations of a dioxin/furan mixture) using a 96-well luminometer.
The mean RLU of the control wells is set to unity. Mean RLUs
of samples are first recorded as fold induction (times back-
ground), which is derived by dividing by the mean RLU of the
solvent (background control). A dioxin/furan standard curve
关toxic equivalents (TEQ) vs. fold induction兴is prepared from the
results of each test, and the slope is used to adjust fold induction
values to TEQs (in ng/g dry weight). The RGS response repre-
sents the integrated CYPlAl induction from all dioxin-like com-
pounds in the extract. Because standard curves have been pre-
pared for the response to benzo(a)pyrene and a mixture of
dioxins and furans, the assay results may be expressed as mi-
crograms of benzo(a)pyrene equivalents, or nanograms of TCDD
TEC per gram dry weight or per liter.
3. References
1. ANDERSON, J.W., S.S. ROSSI, R.H. TUKEY,T.VU& L.C. QUATTROCHI.
1995. A biomarker, P450 RGS, for assessing the induction potential
of environmental samples. Environ. Toxicol. Chem. 14:1159.
2. JONES, J.M. & J.W. ANDERSON. 1999. Relative potencies of PAHs
and PCBs based on the response of human cells. Environ. Toxicol.
Pharmacol. 7:19.
3. ANDERSON, J.W., J.M. JONES,J.HAMEEDI,E.LONG &R.TUKEY. 1999.
Comparative analysis of sediment extracts from NOAA’s Bioeffects
studies by the biomarker, P450 RGS. Mar. Environ. Res. 48:407.
4. ANDERSON, J.W., J.M. JONES,S.STEINERT,B.SANDERS,J.MEANS,D.
MCMILLIN,T.VU&R.TUKEY. 1999. Correlation of CYP1A1
induction, as measured by the P450 RGS biomarker assay, with high
molecular weight PAHs in mussels deployed at various sites in San
Diego Bay in 1993 and 1995. Mar. Environ. Res. 48:389.
5. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1995. Methods 3540 and
3550 in EPA Test Methods for Evaluating Solid Waste, Physical-
Chemical Methods, SW-846, 3rd ed., Update 2B, March, 1995. U.S.
Environmental Protection Agency, Washington, D.C.
6. SAFE, S. 1994. Polychlorinated biphenyls (PCBs): Environmental
impact, biochemical and toxic responses, and implications for risk
assessment. Crit. Rev. Toxicol. 24:87.
7. STEGEMAN, J., M. BROUWER, R.T. DIGIULIO,L.FORLIN, B.A. FOWLER,
B.M. SANDERS & P.A. VAN VELD. 1992. Molecular responses to
environmental contamination: enzyme and proteins systems as in-
dicators of chemical exposure and effect. In R.J. Huggett, R.A.
Kimerle, P.M. Mehrle, Jr. & H.L. Bergman, eds. Biomarkers: Bio-
chemical, Physiological, and Histological Markers of Anthropo-
genic Stress, p. 235. Lewis Publishers, Boca Raton, Fla.
8. POSTLIND, H., T.P. VU, R.H. TUKEY & L.C. QUATTROCHI. 1993.
Response of human CYPl-luciferase plasmids to 2,3,7,8-tetrachlo-
rodibenzo-p-dioxin and polycyclic aromatic hydrocarbons. Toxicol.
Appl. Pharmacol. 118:255.
9. AMERICAN SOCIETY FOR TESTING AND MATERIALS. 2009. Guide for
measuring the presence of planar organic compounds which induce
CYP1A, using reporter gene test systems. E1853M-98, Annual
Book of ASTM Standards, Vol. 11.05. American Soc. Testing &
Materials, W. Conshohocken, Pa.
10. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1999. Method 4425:
Screening Extracts of Environmental Samples for Planar Organic
Compounds (PAHs, PCBs, PCDDs/PCDFs) by a Reporter Gene on
a Human Cell Line, SW 846 Methods, Update IVB. Off. Solid
Waste, U.S. Environmental Protection Agency, Washington, D.C.
11. JONES, J.M. & J.W ANDERSON. 2000. Using the metabolism of PAHs
in a human cell line to characterize environmental samples. Environ.
Toxicol. Pharmacol. 8:119.
4. Bibliography
LEE, R.F. & J.W. ANDERSON. 2005. Significance of cytochrome P450
system responses and levels of bile fluorescent aromatic compounds
in marine wildlife following oil spills. Marine Pollution Bull.
50:705.
CHRISTEN, V., D.M. OGGIER &K.FENT. 2009. A microfilter-plate-based
cytochrome P450 3A activity assay in fish cell lines. Environ.
Toxicol. Chem. 28:2632.
P450 REPORTER GENE RESPONSE (8070)/The P450 RGS Test
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P450 REPORTER GENE RESPONSE (8070)/The P450 RGS Test