Sediment Quality in Puget Sound Year 2 - Center for Coastal ...

have been exposed to one or more of these xenobiotic organic compounds, including dioxins,
furans, planar PCBs, and several polycyclic aromatic hydrocarbons (Jones and Anderson, 1999).
Differences in the ability of the P450 enzyme to metabolize chlorinated and non-chlorinated
compounds allow for differentiation between these classes of compounds in environmental
samples. Since most PAHs are rapidly metabolized, they exhibit a maximum response in 6
hours, at which point the response begins to fade. Chlorinated hydrocarbons (dioxins, furans,
and certain PCBs), on the other hand, do not show a maximum response until 16 hours after
exposure (Jones and Anderson, 2000). The P450 HRGS assay provides an estimate of the
presence of contaminants bound to sediment that could produce chronic andlor carcinogenic
effects in benthic biota and/or demersal fishes that feed in sediments. These tests were run by the
Columbia Analytical Services, Inc. in Vista, CA with solvent extracts prepared by their
laboratory in Kelso, WA.
The details of this test are provided as U.S. EPA Method 4425 (EPA, 1999), Standard Method
8070 by the American Public Health Association (APHA, 1998), and ASTM method E 1853M-
98 by the American Society for Testing and Material (ASTM, 1999). The test uses a transgenic
cell line (1 OIL), derived from the human hepatoma cell line (HepG2), in which the flanking
sequences of the CYP 1A gene, containing the xenobiotic response elements (XREs), have been
stably linked to the firefly luciferase gene (Anderson et al. 1995, 1996). As a result, the enzyme
luciferase is produced in the presence of compounds that bind the XREs.
After removal of debris and pebbles, the sediment sample was homogenized, dried with
anhydrous sodium sulfate, and 20 g of sediment was extracted by sonication with
dichlorometl~ane (DCM), also known as methylene chloride. The extract was carefully
evaporated and concentration under a flow of nitrogen, and exchanged into mixture of
dimetl~ylsulfoxide (DMSO), toluene and isopropyl alcol~ol (2: 1 : 1) to achieve a final volume of 2
mL. The 2 mL extracts were split into two 1 mL vials for testing with the Microtox and P450
HRGS assays. The extraction procedure is well suited for extraction of neutral, non-ionic
organic compounds, such as aromatic and chlorinated hydrocarbons. Extraction of other classes
of toxicants, such as metals and polar organic compounds, is not efficient. DMSO is compatible
with these tests because of its low toxicity and high solubility with a broad spectrum of non-polar
chemicals.
Briefly, a small amount of organic extract of sediment (up to 20 pL), was applied to
approximately one million cells in each well of a 6-well plate with 2 mL of medium. Detection
of enzyme induction in this assay is relatively rapid and simple to measure since binding of a
xenobiotic with the Ah receptor results in the production of luciferase.
After 16 hours of incubation with the extract, the cells are washed and lysed. Cell lysates are
centrifuged, and the supernatant is mixed with buffering chemicals. Enzyme reaction is initiated
by injection of luciferin. The resulting luminescence is measured with a luminometer and is
expressed in relative light units (RLUs). A solvent blank (using a volume of solvent equal to the
sample's volume being tested) and reference toxicants (TCDD, dioxinlfuran mixture, B[a]P) are
used with each batch of samples.
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