Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays.
Sediments may be the ultimate sink for persistent (xeno-)estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-CALUX assay was more sensitive to 17beta-estradiol (E2) than the recombinant yeast screen, with an EC50 of 6 pM E2 compared to 100 pM in the yeast screen. Yeast cells were unable to distinguish the anti-estrogens ICI 182,780 and (4-hydroxy)tamoxifen, which were agonistic in the yeast. Acetone-soluble fractions of hexane/acetone extracts of sediments showed higher estrogenic potency than hexane-soluble extracts in the ER-CALUX assay. Sediments obtained from industrialized areas such as the Port of Rotterdam showed the highest estrogenic potency of the 12 marine sediments tested (up to 40 pmol estradiol equivalents per gram sediment). The estrogenic activity of individual chemicals that can be found in sediments including: alkylphenol ethoxylates and carboxylates; phthalates; and pesticides, was tested. Increasing sidechain length of various nonylphenol ethoxylates resulted in decreased estrogenic activity. Of the phthalates tested, butylbenzylphthalate was the most estrogenic, though with a potency approximately 100,000 times less than E2. The organochlorine herbicides atrazine and simazine failed to induce reporter gene activity. As metabolic activation may be required to induce estrogenic activity, a metabolic transformation step was added to the ER-CALUX assay using incubation of compounds with liver microsomes obtained from PCB-treated rats. Results indicate that metabolites of E2, NP and bisphenol A were less active than the parent compounds, while metabolites of methoxychlor were more estrogenic following microsomal incubations.
Legler
Juliette
J
Division of Toxicology, Wageningen University, The Netherlands. juliette.legler@ivm.vu.nl
Dennekamp
Martine
M
Vethaak
A Dick
AD
Brouwer
Abraham
A
Koeman
Jan H
JH
van der Burg
Bart
B
Murk
Albertinka J
AJ
D000818:Animals; D001280:Atrazine / Q000009:adverse effects*; D001681:Biological Assay; D004785:Environmental Pollutants / Q000009:adverse effects*; D004968:Estrogens, Non-Steroidal / Q000009:adverse effects*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D019015:Geologic Sediments / Q000737:chemistry*; D006540:Herbicides / Q000009:adverse effects*; D008156:Luciferases / Q000096:biosynthesis; D011078:Polychlorinated Biphenyls / Q000009:adverse effects*; D051381:Rats; D011960:Receptors, Estrogen / Q000187:drug effects*; D014874:Water Pollutants, Chemical / Q000009:adverse effects*; D015003:Yeasts
D004785:Environmental Pollutants; D004968:Estrogens, Non-Steroidal; D006540:Herbicides; D011960:Receptors, Estrogen; D014874:Water Pollutants, Chemical; D011078:Polychlorinated Biphenyls; D008156:Luciferases; D001280:Atrazine