Rapid Assessment of Induced Cytochrome P4501a Protein and Catalytic Activity in Fish Hepatoma Cells Grown in Multiwell Plates: Response to Tcdd, Tcdf, and Two Planar PCBS

Abstract

Abstract-Induction of cytochrome P450 1A1 (CYP1A1) in cultured cells can be used to determine taxon-specific relative potencies of Ah receptor agonists. This report describes optimized methods for growth and treatment of PLHC-1 fish hepatoma cells in multiwell plates, in situ analysis of ethoxyresorufin O-deethylase (EROD) activity, and measurement of CYP1A protein by immunoblotting of cell lysates. EROD activity was undetectable (Ͻ1 pmol min Ϫ1 mg Ϫ1) in untreated or dimethyl sulfoxide-treated cells, but was highly induced (up to 150 pmol min Ϫ1 mg Ϫ1) in cells exposed to Ah receptor agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), or planar chlorobiphenyls (CB). Addition of exogenous NADPH was not required for measurement of EROD activity in PLHC-1 cells. As inducers of EROD activity, TCDD, TCDF, 3,3Ј,4,4Ј,5-pentachlorobiphenyl (CB-126), and 3,3Ј,4,4Ј-tetrachlorobiphenyl (CB-77) differed both in potency and in apparent efficacy (maximal level of induced activity). In each case, EROD induction was biphasic, with stronger induction at lower concentrations and an attenuated response at higher concentrations. In contrast, the content of immunodetectable CYP1A protein increased monotonically with dose of CB, and the maximum level achieved was similar for all inducers. The discrepancy in results obtained for EROD activity versus CYP1A protein may result from inhibition or inactivation of catalytic function at high concentrations of inducer. By reducing peak EROD values, this inhibition leads to lower apparent EC50 values and thus the overestimation of relative potencies or toxic equivalency factors (TEFs) for many inducers. These studies demonstrate the necessity of measuring both EROD activity and immunodetectable CYP1A protein for the accurate assessment of CYP1A induction and relative potencies in cultured cells.

Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife.

“…It The most extensively used biological in vitro response studied so far is the induction of the CYPIAJ gene. CYPJAJ gene induction has been measured by monitoring changes in mRNA (165,166), protein levels (66,167), and/or enzyme activity [i.e., EROD or aryl hydrocarbon hydroxylase (AHH)] (168,169).…”

Organochlorine Chemical Residues in Fish from the Mississippi River Basin, 1995

“…HCB is shorter-lived (Villanueva et al 1974) and much less toxic (Jarvinen and Ankley 1999) than DDT and most other persistent organochlorine compounds; however, commercial formulations once contained toxic impurities, including polychlorinated dibenzo-pdioxins and -dibenzofurans (Villanueva et al 1974). In addition, HCB has a low level of dioxin-like activity (about 0.0001-0.001 relative to 2,3,7,8-TCDD- Hahn et al 1996;Sinclair et al 1997), and may therefore contribute to toxicity in combination with other polyhalogenated hydrocarbons. In 1995, HCB residues were detected (about 0.01 g/g) in only four samples (2%) from three sites (7%, Table 2): NCBP Stations 24 (Ohio R. at Marietta, OH), 76 (Mississippi R. at Memphis), and 23 (Kanawha R. at Nitro, WV).…”

Characterization of resistance to halogenated aromatic hydrocarbons in a population of Fundulus heteroclitus from a marine superfund site

“…The hepatocytes were plated in 96 well plates at I x 1 0 cells / well and allowed to attach overnight. The hepatocytes were then exposed to TCDD 0.001 -30 nM or BNF 1 -10,000 nM in DMSO or DMSO alone for 24 hours, using methods described previously Hahn et al, 1996. Four replicate wells were exposed at each concentration.…”