Effects-directed analysis of sediments from polluted marine sites in Norway.

The environmental status of two polluted marine sites in Norway was investigated by a combination of target chemical analysis and effect-directed analysis (EDA). The two selected sites, the Grenland area and Oslo harbor, in addition to two reference sites, were classified according to the Norwegian environmental classification system based upon results of the target chemical analyses. The polluted sites were characterized by high levels of metals, polycyclic aromatic hydrocarbons (PAH), and polychlorinated biphenyls (PCB). High levels of organotin compounds were also detected in Oslo harbor. The aryl hydrocarbon receptor (AhR) agonist activity in extracts of sediments from marine sites close to Oslo, Oslo harbor, and Grenland were investigated using the CALUX (chemical-activated luciferase expression) assay, which showed elevated levels of activity. As expected from the history of dioxin release into the Grenland area, the results were highest in this area. The presence of estrogen receptor (ER) and androgen receptor (AR) antagonists was also detected in the sediment extracts. Following fractionation of the sediment extracts, EDA was used to tentatively identify the AhR agonists. The compounds responsible for AhR agonist activity in samples from Oslo harbor were isolated in fraction 13, and to a lesser extent in fractions 9-11. In Grenland, the main activity was found in the more polar fractions, namely fractions 14-18. The AhR agonists identified in Oslo harbor were mainly PAH, while in the Grenland area the compounds identified were mainly nitrogen/oxygen-containing polyaromatic compounds (N/O-PAC).

Estrogenicity of alkylphenols and alkylated non-phenolics in a rainbow trout (Oncorhynchus mykiss) primary hepatocyte culture.

Alkylphenols act as estrogen mimics by binding to and transactivating estrogen receptors (ERs) in fish. In the present study, activation of ER-mediated production of the estrogenic biomarker vitellogenin (vtg) in a primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes was used to construct a structure-activity relationship for this ubiquitous group of aquatic pollutants. The role of alkyl chain length and branching, substituent position, number of alkylated groups, and the requirement of a phenolic ring structure was assessed. The results showed that most alkylphenols were estrogenic, although with 3-300 thousand times lower affinity than the endogenous estrogen 17beta-estradiol. Mono-substituted tertiary alkylphenols with moderate (C4-C5) and long alkyl chain length (C8-C9) in the para position exhibited the highest estrogenic potency. Substitution with multiple alkyl groups, presence of substituents in the ortho- and meta-position and lack of a hydroxyl group on the benzene ring reduced the estrogenic activity, although several estrogenic alkylated non-phenolics were identified. Co-exposures with the natural estrogen 17beta-estradiol led to identification of additional estrogenic compounds as well as some anti-estrogens. A combination of low affinity for the ER and cytotoxicity was identified as factors rendering some of the alkylphenols non-estrogenic in the bioassay when tested alone.

Gill EROD in monitoring of CYP1A inducers in fish: a study in rainbow trout (Oncorhynchus mykiss) caged in Stockholm and Uppsala waters.

The gill filament 7-ethoxyresorufin O-deethylase (EROD) assay was evaluated as a monitoring tool for waterborne cytochrome P4501A (CYP1A) inducers using rainbow trout (Oncorhynchus mykiss) caged in urban area waters in Sweden. To compare the CYP1A induction response in different tissues, EROD activity was also analyzed in liver and kidney microsomes. Immunohistochemistry was used to localize CYP1A protein in gill and kidney. In two separate experiments fish were caged at sites with fairly high expected polyaromatic hydrocarbon (PAH) contamination. In the first experiment, gill EROD activities were analyzed in fish exposed for 1-21 days in a river running through Uppsala. The reference site was upstream of Uppsala. In the second, gill, liver and kidney EROD activities were analyzed in fish exposed for 1-5 days in fresh or brackish waters of Stockholm and in a reference lake 60km north of Stockholm. Fish exposed for 5 days followed by 2 days of recovery in tap water in the laboratory were also examined. The gill consistently showed a higher EROD induction compared with the liver and the kidney. After 1 day of caging, gill EROD activity was markedly induced (6-17-fold) at all sites examined. Induction in gill was pronounced (5-7-fold) also in fish caged at the reference sites. In the 21-day exposure study gill EROD activity remained highly induced throughout the experiment (26-fold at most) and the induced CYP1A protein was exclusively confined to the gill secondary lamellae. In the 5-day exposure experiment, EROD activity peaked after 1 day and then declined in both gill and liver, while CYP1A immunostaining in the gill remained intense over the 5-day period. In the kidney, CYP1A staining was weak or absent. We conclude that gill EROD activity is a more sensitive biomarker of exposure to waterborne CYP1A inducers than EROD activity in liver and kidney.