705 Environmental Toxicology and Chemistry, Vol. 23, No. 3, pp. 705–711, 2004  2004 SETAC Printed in the USA 0730-7268/04 $12.00 + .00 ESTROGENIC POTENTIAL OF HALOGENATED DERIVATIVES OF NONYLPHENOL ETHOXYLATES AND CARBOXYLATES NATA ` LIA GARCI ´ A-REYERO,² V ANESSA REQUENA,² M IRA PETROVIC,‡ BIRGIT FISCHER,§ PETER-DIEDRICH HANSEN,§ ALFREDO DI ´ AZ, FRANCESC VENTURA, DAMIA ` BARCELO ´ ,‡ and BENJAMIN PIN ˜ A*² ²Institut de Biologia de Barcelona, Consejo Superior de Investigaciones Cientı ´ficas, Jordi Girona, 18, 08034 Barcelona, Spain ‡Institut de Investigacions Quı ´miques i Ambientals, Consejo Superior de Investigaciones Cientı ´ficas, Jordi Girona, 18, 08034 Barcelona, Spain §Technische Universita ¨t Berlin, Department of Ecotoxicology, Franklimstrasse 29, D-10589 Berlin, Germany AGBAR, Aigu ¨es de Barcelona, P. Sant Joan 39, 08009 Barcelona, Spain ( Received 11 March 2003; Accepted 25 July 2003) Abstract—Halogenated derivatives of nonylphenol and of its alkylates are generated during drinking water disinfection and treatment procedures. In this paper we analyze the potential of these compounds to interact with the estrogen receptor and to activate hormone- regulated gene promoters. We used the recombinant yeast assay (RYA) and the human breast cancer cell MCF7 proliferation assay for both estrogenic and antiestrogenic activities and the enzyme-linked receptor assay to examine in vitro binding to the receptor. Many nonylphenol derivatives were very weak estrogens in our functional tests when compared to nonylphenol while retaining a substantial affinity for the estrogen receptor in vitro. Antiestrogenicity tests demonstrated that brominated nonylphenol and most of the carboxylated compounds studied here behaved as estrogenic antagonists in the RYA. We also detected an increasedcytotoxicity for the carboxylated derivatives in both yeast and mammalian cells. We conclude that derivatization may mask the apparent estrogenicity of nonylphenol, but the resulting compounds still represent a potential hazard since they are still able to bind the estrogen receptor and to influence the physiological response to estrogens. Our results also illustrate the advantage of combining different methods to assay estrogenicity of unknown substances. Keywords—Endocrine disruptors Recombinant yeast assay Enzyme-linked receptor assay Nonylphenol derivatives INTRODUCTION Vertebrate estrogen receptors can interact with a variety of chemical compounds with little or no obvious chemical sim- ilarity with the natural ligands [1]. As a consequence, many of the numerous new compounds created yearly by the chem- ical industry have the potential to disrupt the delicate hormonal balance of humans and nontarget organisms exposed to them. This risk is especially relevant for the aquatic fauna, exposed to urban and industrial discharges containing many of these potential hormone disruptors. Nonylphenol derivatives are widely used as industrial clear- ing agents in processes requiring defoaming, de-emulsifica- tion, dispersion, or solubilization. The degradation of these compounds is the major environmental source of nonylphenol (NP), which is found in g/L amounts in impacted rivers and estuaries [2]. Although originally considered relatively non- toxic, NP has demonstrated to be estrogenic both in laboratory tests and in the field [1,3–5]. Much less information exists on the estrogenicity of NP derivatives, although it is supposed to be much lower than that of the parental compound [6,7]. In this work we focused on nonylphenol ethoxylates (NPEO) and carboxylates (NPEC) as well as on their chlorinated and bro- minated derivatives (ClNPXs and BrNPXs, respectively). These halogenated derivatives are produced during tap water treatment procedures, such as chlorine disinfection in the pres- ence of bromide ions. Amounts ranging from ppb to ppm of these compounds were recently reported in prechlorinated wa- * To whom correspondence may be addressed (bpcbmc@cid.csic.es). ter and sludge from the Barcelona drinking water treatment plant, although they appeared to be absent (at the sub-ppb level of detection) from the final tap water [8]. In this work we attempt the study of the potential estro- genicity of nonylphenol derivatives by combining three in vitro methods that monitor three distinct aspects of the estrogen response in vertebrate cells. The recombinant yeast assay (RYA) uses genetically engineered yeast cells to reproduce the natural pathway of genetic control by estrogens in vertebrate cells [1,9,10]; it has the advantage of its simplicity and a high throughput. The MCF-7 proliferation assay (E-test) is based on the property of estrogens to stimulate proliferation of breast cancer cells in culture [10,11], although the underlying genetic mechanism is not fully understood yet. The enzyme-linked receptor assay (ELRA) [11] measures the capacity of a given compound to compete with 17--estradiol (estradiol) for bind- ing to the human estrogen receptor in vitro. The combination of these assays allows one to estimate the affinity of different compound for the human estrogen receptor as well as to ex- plore the possible physiological consequences of this inter- action. They can indicate whether a given compound would contribute to the estrogenic response (agonists), inhibit the response to the natural estrogens (antagonists or antiestrogens), or a combination of both (partial agonists). Our results indicate that derivatization affected estrogenicity to a much greater extent than it affects binding to the receptor. This leads to the unexpected result that nonylphenol derivatization (particularly bromination and carboxylation) may have the potential for transforming the estrogenic parental compound into an anti- estrogen.