Biological Evaluation of the Pollution of Rivers Flowing into Tokyo Bay with the 7-Ethoxycoumarin O-deethylase(ECOD) Activity Induced by River Sediment Extracts in HepG2 Cells.

Abstract

The extent of pollution in the rivers flowing into Tokyo Bay was studied comparatively using river sediment extracts in an assay system based on the arylhydrocarbon receptor (AhR)-dependent induction of 7-ethoxycoumarin O-deethylase (ECOD) activity in HepG2 cells. The sampling points of river sediment were as follows: Namamugi on the Tsurumi River, Rokugo on the Tama River, Hirai, Senjyu and Funado on the Ara River, Baraki on the Edo River, and Makuhari on the Hanami River (Fig. 1). The Tsurumi River (Namamugi) located in the middle of Keihin Industrial District was considered most polluted, followed by the Ara River (Hirai). The river sediment collected at Baraki on the Edo River was least polluted. The extracts of highly polluted river sediments sampled at Namamugi and Hirai exhibited a reverse U-shaped dose-response curve, for which polycyclic aromatic hydrocarbons (PAHs) might be mainly responsible, in a range of non-toxic concentrations. The pollution of river sediment collected at Namamugi was roughly estimated to be 2 to 20 µg as PAHs/g sediment by consulting the data of Nakama et al.1) and 0.2 to 2 µg as PAHs/g was assigned to the river sediment of Hirai.

Possible Involvement of Chlorinated Ethylenes in Arylhydrocarbon Receptor-Related Induction of Cytochrome P4501A1 (CYP1A1) in Human Hepatoma HepG2 Cells

“…Ethoxycoumarin O-Deethylase (ECOD) Activity ---The measurement of ethoxycoumarin Odeethylase (ECOD) activity was performed as described previously. [12][13][14] HepG2 cells treated with CEs were incubated in 0.9 ml of Minimum Essential Medium (GIBCO™/Invitrogen) containing HEPES (final concentration, 12 mM), glucose (final 30 mM) and 7-ethoxycoumarin (final 1 mM) at 37°C for 3 hr, and the reaction was stopped with ice-cold acetone. After centrifugation of the reaction mixture, the pellet was used for determination of the protein concentration with Lowry's method.…”

Biological Evaluation of Pollution in the Sagami River Using the 7-Ethoxycoumarin O-Deethylase Activity Induced by River Sediment Extracts in HepG2 Cells

“…Determination of ECOD Activity ---The determination of ECOD activity was performed as described previously. 5) A Type F-3000 fluorescence spectrophotometer (Hitachi, Japan) was used for fluorescence spectrophotometry.…”

“…The rivers flowing into Tokyo Bay were studied comparatively by examining the extent of pollution with AhR-agonists such as PCDDs/Fs, polychlorinated biphenyls (PCBs), and PAHs using surface sediment extracts sampled at points near their estuaries. 5,6) Among them, the Tsurumi River ranked first in terms of potential to induce AhR-dependent 7-ethoxycoumarin Odeethylase (ECOD) activity. To determine the cause of the heavy pollution at the estuaries, the pollution of the Tsurumi River was studied longitudinally, showing the primary effects of nearby industrial complexes.…”

Biological Evaluation of the Pollution of the Tsurumi River with 7-Ethoxycoumarin O-Deethylase Activity Induced by River Sediment Extracts in HepG2 Cells.

“…[2][3][4] Although CYP in cultured cells cannot generally be induced, human hepatoma cell line HepG2 retains this feature and has been applied to a bioassay for environmental pollutants in combination with 7-ethoxycoumarin as a substrate with satisfactory sensitivity. 5) In a previous study, 6) surface sediment collected at Namamugi on the Tsurumi River estuary was reported to be the most polluted among those collected from several rivers scattered in the Tokyo metropolitan area. This conclution was based on result from a bioassay measuring 7-ethoxycoumarin Odeethylase (ECOD) activity in HepG2 cells.…”