A novel method for measuring aromatase activity in tissue samples by determining estradiol concentrations H. Tinwell, a * J. B. Rascle, a S. Colombel, a I. Al Khansa, a A. Freyberger b and R. Bars a ABSTRACT: Increasing scrutiny of endocrine disrupters has led to changes to European pesticide and biocide legislation and to the introduction of the Endocrine Disrupter Screening Program by the US EPA. One element of endocrine disrupter identification is to determine its effects on aromatase, but most available assays are limited as they depend on tritiated water production to indicate enzyme activity. Whilst acceptable for determining aromatase effects using a cell‐free approach, this method is unreliable for cell or tissue‐based investigations as other cytochrome P‐450 isoenzyme activities can similarly produce tritiated water and consequently confound interpretation of the aromatase data. To address this lack of specificity an assay directly measuring the final estrogen product by incubating rat tissue protein with testosterone and measuring the resultant estradiol concentration was developed. Using this approach we demonstrated marked increases in enzyme activity in pregnant rat ovary samples and dose‐related inhibitions when incubating non‐pregnant rat ovary samples with known aromatase inhibitors. Hepatic aromatase activity was investigated using our method and by tritiated water production with microsomes from rats dosed with the antiandrogen 1,1‐dichloro‐2,2‐bis(4 chlorophenyl)ethane. Additional cytochrome P‐450s were also measured. Treatment‐related increased tritiated water production and general hepatic enzyme activity were recorded but estradiol was not increased, indicating that the increased tritiated water was due to general enzyme activity and not aromatase activity. A simple and specific method has been developed that can detect aromatase inhibition and induction, which when applied to tissue samples, provides a means of generating relevant animal data concerning chemical effects on the aromatase enzyme. Copyright © 2011 John Wiley & Sons, Ltd. Keywords: aromatase; endocrine disruption; estradiol; liver; 1,1‐dichloro‐2,2‐bis(4 chlorophenyl)ethane (DDE) INTRODUCTION Concern that alterations to the endocrine system by chemicals can result in adverse health effects in the human population and wildlife has led to increased scrutiny in both Europe and the USA of endocrine disrupters. For example, in the USA, the Environmental Protection Agency (EPA) has launched an Endocrine Disrupter Screening Program (EDSP; http://www. epa.gov/endo) to investigate the endocrine disrupting potential of registered pesticides. Furthermore, recent European legisla- tive changes have led to a more stringent regulation of certain chemicals, particularly pesticides (Plant Protection directive EC no. 1107/2009) and biocides (biocides directive 98/8/EC), with endocrine disrupting properties. Aromatase (Cyp19) is a key enzyme in steroid hormone biosynthesis, being responsible for the conversion of androgens to estrogens (Mendelson et al., 1985; Thompson and Siiteri, 1974). It is found predominantly in the ovaries but also in mammary tissue, testis and brain and, in humans, in the placenta. Increased intrinsic activity of this enzyme has been causally linked to mammary, uterine and adrenal tumors (Bulun et al., 1997; Brodie and Njar, 2000) and as a result a number of pharmaceutical compounds (e.g. formestane and anastrozole) have been developed to specifically inhibit aromatase activity. However, as aromatase is important in maintaining the homeostatic balance between androgens and estrogens, unwarranted inhibition or stimulation of this enzyme by xenobiotics could be considered as endocrine disruption. For example, the chlorotriazine herbicide atrazine has been shown to increase aromatase activity (Sanderson et al., 2002), which is thought to be responsible for treatment‐related mammary tumors and advanced reproductive senescence in female rats (Cooper et al., 2007). Effects on aromatase activity have also been suggested for some fungicides such as vinclozolin (Sanderson et al., 2002) as well as biocides such as triphenyltin and tributyltin (Heidrich et al., 2001; Saitoh et al., 2001). Such is the importance of aromatase in sexual differentiation, fertility and carcinogenesis that the assessment of the effects of xenobiotics on this enzyme is obligatory for the EPAs EDSP. Several in vitro methods are available to evaluate aromatase activity using either a cell‐free approach such as human placenta microsomes (Vinggaard et al., 2000 and is the method *Correspondence to: H. Tinwell, Bayer SAS, Bayer CropScience, Research Toxicology, Sophia Antipolis, France. E-mail: helen.tinwell@bayer.com a Bayer SAS, Bayer CropScience, Research Toxicology, Sophia Antipolis, France b Pathology and Clinical Pathology, Wuppertal‐Elberfeld, Germany J. Appl. Toxicol. 2011; 31: 446–454 Copyright © 2011 John Wiley & Sons, Ltd. Research Article Received: 10 September 2010, Revised: 12 October 2010, Accepted: 13 October 2010 Published online in Wiley Online Library: 24 January 2011 (wileyonlinelibrary.com) DOI 10.1002/jat.1623 446