Cytochrome P4501A Induction and Porphyrin Accumulation in PLHC-1 Fish Cells Exposed to Sediment and Oil Shale Extracts

Abstract

The present study describes the use of a fish hepatoma cell line (PLHC-1) in monitoring the biological effects of sediments collected from recipient waters of the oil shale industry. Sampling sites were located in River Purtse and River Kohtla in northeast Estonia. The effects of pure oil shale on the PLHC-1 cells were also studied. The cells were exposed to n-hexane-extracted samples in 48-well plates for 24 h, and 7-ethoxyresorufin O-deethylase (EROD) activity, total protein, and porphyrin content were measured in the exposed cells. Polycyclic aromatic hydrocarbon (PAH) contents in the samples were measured by high-performance liquid chromatography (HPLC). All the sediment and oil shale samples induced CYP1A activity and led to porphyrin accumulation in the cells. The most potent inducers were the sediments collected near the oil shale processing plants (site Lüganuse in River Purtse and Kohtla in River Kohtla), as well as those at the most downstream site in River Purtse (Purtse). These samples possessed high total PAH contents, ranging from 4,270 to nearly 150,000 microg/kg dry sediment. The presence of other lipophilic organic contaminants in the samples was not determined in this study. Both EROD activity and porphyrin content exhibited biphasic induction curves, and the ED(50)(1) values for EROD activity were lower than the ED(50)s for porphyrin content. 2,3,7, 8-Tetrachlorodibenzo-p-dioxin induction equivalents (TCDD-EQs) calculated from EROD induction potencies correlated well with total PAHs (r(2) = 0.827 and p = 0.003 for log-transformed data) and also with individual PAHs. TCDD-EQs for porphyrin content did not correlate significantly with total PAHs (log-log r(2) = 0.785, p = 0. 116). The biological potency and PAH contamination of the samples showed the same rank order, except at Lüganuse, where sediment extracts induced CYP1A and porphyrins more than could have been expected based on PAH contents. Bioassay-derived induction EQs (normalized to dibenz(a,h)anthracene) were 20- to 3,200-fold greater than EQs calculated from the concentrations of five PAHs, suggesting important contributions from other compounds or nonadditive effects. The PLHC-1 cells proved to be a sensitive bioanalytical tool for sediments contaminated with PAH-type pollutants in the oil shale processing area. We suggest further use of this bioassay in screening and monitoring waters with similar background of pollution as in northeast Estonia.

Mechanisms of action for aryl hydrocarbon receptor ligands in the PLHC-1 cell line

“…These cells have been used for investigations of cytotoxicity (Babich et al 1991b;Ryan and Hightower 1994;Bruschweiler et al 1995;Huuskonen et al 1998a;Fent and Batscher 2000) and effects of AHR ligands (Bruschweiler et al I996a;Bruschweiler et al 1996b;Celander et al 1996;Celander et al 1997;Villeneuve et al 1997;Huuskonen et al 1998b;Huuskonen et al 1998c;Smeets et al 1999;Huuskonen et al 2000). PLHC-I cells show induction of CYPIA (an AHR-mediated response) in response to AHR agonists (Hahn et al 1993;Hahn et al 1996a;Hahn et al 1996b;Hestermann et al in press).…”

In vitro cellular responses in the RTG‐2 cell line to complex mixtures of dioxins and dioxin‐like PCDDs, PCDFs and PCBs

“…Similar differences were obtained in this study using the RTG-2 cell line. H4IIE and PLHC-based in vitro EROD-TEQ bioassays were in a similar range to RTG-2 cells for detecting PBT pollutants in complex matrices by comparing EROD-TEQ/ chemoanalytical TEQ values [for H4IIE cell lines 1.6 (Behnisch et al, 2002) and 461 (Roy et al, 2002), and for RTG-2 cell line 1.2 (this work)] or by comparing regression values [for PLHC cell line 0.85 (Huuskonen et al, 2000), for rat hepatocytes 0.75 (Till et al, 1997) and for RTG-2 cell line 0.88 (this work)]. Although more testing samples are needed in order to recommend the EROD-RTG-2 bioassay, the use of this cell line was found to be suitable as an estimate for the level of PCDDs/PCDFs and DL-PCBs in the waste samples analysed in this work.…”

“…In vitro CYP1A induction has also been used to assess the level of dioxin equivalents in a number of environmental samples using rat hepatome H4IIE cell line (Hanberg et al, 1991;Tillitt et al 1991 et al., 2002) and fish hepatome PLHC-1 (Huuskonen et al, 2000;Traven et al, 2008)) cell line. In all instances, there were EROD-TEQ values determined in the bioassay that were higher and lower than the ones expected from the chemical analysis of PCDDs/ PCDFs and PCBs.…”

Assessment of metabolic capabilities of PLHC-1 and RTL-W1 fish liver cell lines

“…M2, M3 and M4 could not be identified. No -not studied, nd not detected, 4-NP 4-nitrophenol, CDNB 1-chloro-2,4-dinitrobenzene, FIH freshly isolated hepatocytes, GST glutathione-S-transferase, HAQO 4-hydroxyamino-quinoline-1-oxide, NQO 4-nitroquinoline-1-oxide a Bols et al (1999) b Celander et al (1996) c Cravedi et al (1996) d Hahn et al 1996 e Huuskonen et al (2000) f Nabb et al (2006) g Nehls and Segner (2001) h Sadar and Andersson (2001) i Smeets et al (1999) j Sturm et al (2001) LA hydroxylation product was detected in PLHC-1 intact cells. RTL-W1 cells metabolised LA in a similar manner to PLHC-1 cells, but the metabolic rate was lower (data not shown).…”