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.
11
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