Jack W. Anderson

Using the metabolism of PAHs in a human cell line to characterize environmental samples.

P450 reporter gene system (RGS) is an in vitro assay to detect compounds that activate the Ah receptor and induce cytochrome P450 (CYP1A1). This system utilizes a human cell (101L) stably transfected with a luciferase reporter downstream of human CYP1A1 promoter sequences. When CYP1A1-inducing compounds are present, luciferase is produced as well as endogenous CYP1A1 enzymes. Polycyclic aromatic hydrocarbons (PAHs) are more readily degraded than chlorinated compounds including dioxins, furans, and coplanar polychlorinated biphenyls (PCBs). PAH-induced luciferase production begins to decrease between 6 and 16 h, while chlorinated compounds produce a more sustained response. Individual and mixtures of CYP1A1-inducing compounds were tested at both 6 and 16 h. Extracts of soils containing both PAHs and dioxins were also tested, before and after cleanup to remove PAHs. Results indicate that RGS testing at 6 and 16 h is a promising tool to differentiate between PAHs and chlorinated hydrocarbons often co-occurring in environmental samples.

Application of P450RGS (reporter gene system) as a bioindicator of sediment PAH contamination in the vicinity of Incheon Harbor, Korea

Sixty seven sediment samples were collected from Kyeonggi Bay, Korea, including the mouth of Han River, Incheon Harbor, the Namdong industrial complex, and the open sea. Collections were conducted in December, 1995 and samples were maintained frozen (-20 °C) until analysis. Dichloromethane extracts were analysed for their content of CYP1A1-inducing compounds with a P450RGS (reporter gene system) assay, and for polycyclic aromatic hydrocarbons (PAHs). Sediment samples were also analysed for organic carbon (OC) content and grain size, to aid in evaluating the relationship between contamination and physical nature of the sediments. The responses of the P450RGS assay to the sediment extracts were expressed as μg of benzo[a]pyrene toxic equivalents per g dry weight (μg g-1 BaPTEQ), and these values correlated well (r2 = 0.624 with total PAHs. BaPTEQ values were also highly correlated with the OC content of the sediments. The determination of P450RGS BaPTEQ is a useful tool, because it is both a rapid and inexp...Sixty seven sediment samples were collected from Kyeonggi Bay, Korea, including the mouth of Han River, Incheon Harbor, the Namdong industrial complex, and the open sea. Collections were conducted in December, 1995 and samples were maintained frozen (-20 °C) until analysis. Dichloromethane extracts were analysed for their content of CYP1A1-inducing compounds with a P450RGS (reporter gene system) assay, and for polycyclic aromatic hydrocarbons (PAHs). Sediment samples were also analysed for organic carbon (OC) content and grain size, to aid in evaluating the relationship between contamination and physical nature of the sediments. The responses of the P450RGS assay to the sediment extracts were expressed as μg of benzo[a]pyrene toxic equivalents per g dry weight (μg g(-1) BaPTEQ), and these values correlated well (r(2) = 0.624 with total PAHs. BaPTEQ values were also highly correlated with the OC content of the sediments. The determination of P450RGS BaPTEQ is a useful tool, because it is both a rapid and inexpensive means of assessing the potential toxicity of organic compounds in environmental sediment samples. These values represent an estimate of the levels of compounds in the sediment that are potentially available to organisms through chronic exposure to pore water or ingestion of benthic species. We believe BaPTEQ values are more useful than tables of specific PAH concentrations, if the purpose of the investigation is to either obtain a rapid screening of an area or to develop some form of ecological or human health risk assessment.

Comparative analysis of sediment extracts from NOAA's bioeffects studies by the biomarker, P450 Reporter Gene System

Abstract Sediment samples collected from nine coastal and estuarine areas have been analyzed to determine response of a biomarker, P450 Reporter Gene System (RGS). This biomarker, using a transgenic cell line, detects the presence and potency of organic compounds that typically induce the CYP1A gene. Previous investigations have shown that sediments containing chemicals which produce a response of 60 or more μg/g of benzo[a]pyrene equivalents (B[a]PEq) also exhibited a degraded community structure. RGS data from all study areas combined (527 samples) show that the mean response is 22.7 μg/g B[a]PEq, and the mean upper 99% confidence interval is 32. We believe that sediments exhibiting B[a]PEq values of 32–60 are possibly contaminated to the extent that effects on benthic organisms may be observed, and those producing an RGS response of 60 and greater are likely to contain levels of chemicals that will produce chronic toxicity. The majority of the stations investigated produced responses below 32 μg/g B[a]PEq. No samples from coastal southern California produced responses greater than 39. However, samples producing induction over 60 μg/g B[a]PEq were: 50%, San Diego Bay; 16%, Delaware Bay; 8%, Sabine Lake; 4%, Northern Puget Sound; and 3%, Charleston Harbor. A strong statistical correlation was observed between the RGS responses and high molecular weight polycyclic aromatic hydrocarbon concentrations in the sediments from several of the study areas. Of the 2694 km2 encompassed in the surveys, only 0.42% of the area exhibited responses above 32 μg/g B[a]PEq, which represented 11.4 km2. This assay is useful in identifying high levels of toxic and carcinogenic compounds in the sediments and predicting the potential impact of these chemicals on the biological community.

Application of P450 reporter gene system (RGS) in the analysis of sediments near pulp and paper mills

Cytochrome P4501A (CYP1A) induction in fish and other animals has been reported following exposure to pulp and paper mill effluent. Dioxins and furans as well as polycyclic aromatic hydrocarbons (PAH) are known inducers of CYP1A and have been found in sediments near pulp and paper mills. Retene (7-isopropyl-1-methylphenanthrene), an alkyl-substituted phenanthrene, has been recently associated with effluent and found to induce CYP1A in fish. This study utilized an in vitro assay, P450 Reporter Gene System (RGS), to assess the transcriptional activation of human CYP1A by retene after short (6 h) and long (16 h) exposures. Retene was as potent as benzo[a]pyrene in inducing RGS, but was not as readily biotransformed by the cells. Extracts of sediments collected near a pulp and paper mill were analysed, and RGS-derived toxic equivalencies (TEQ) were strongly correlated with Chemical TEQ analysis of dioxins and furans determined by EPA Method 8290 using high-resolution gas chromatography/mass spectrometry. RGS 6-h responses indicated the presence of PAH in the extracts, which was confirmed by GC/MS analysis. Retene was detected at considerably higher concentrations than other PAH. These data support the use of the RGS assay to detect the presence of CYP1Ainducing compounds, including retene as well as dioxins and furans, in sediments near pulp mills.Cytochrome P4501A (CYP1A) induction in fish and other animals has been reported following exposure to pulp and paper mill effluent. Dioxins and furans as well as polycyclic aromatic hydrocarbons (PAH) are known inducers of CYP1A and have been found in sediments near pulp and paper mills. Retene (7-isopropyl-1-methylphenanthrene), an alkyl-substituted phenanthrene, has been recently associated with effluent and found to induce CYP1A in fish. This study utilized an in vitro assay, P450 Reporter Gene System (RGS), to assess the transcriptional activation of human CYP1A by retene after short (6 h) and long (16 h) exposures. Retene was as potent as benzo[a]pyrene in inducing RGS, but was not as readily biotransformed by the cells. Extracts of sediments collected near a pulp and paper mill were analysed, and RGS-derived toxic equivalencies (TEQ) were strongly correlated with Chemical TEQ analysis of dioxins and furans determined by EPA Method 8290 using high-resolution gas chromatography/mass spectrometry. RGS 6-h responses indicated the presence of PAH in the extracts, which was confirmed by GC/MS analysis. Retene was detected at considerably higher concentrations than other PAH. These data support the use of the RGS assay to detect the presence of CYP1Ainducing compounds, including retene as well as dioxins and furans, in sediments near pulp mills.