A Practical Approach, Second Edition=Ronald D. Ho.pdf

502 DEVELOPMENTAL REPRODUCTIVE TOXICOLOGY: A PRACTICAL APPROACH, SECOND EDITIONparticular test chemical has an effect on aromatase activity through one of a number of promotersites, as opposed to other cell lines that do not possess multiple promoters.The approach employed to evaluate whether a particular chemical can disrupt the regulation ofaromatase activity depends upon a number of factors. Chemicals that inhibit aromatase activity byacting as suicide substrates can be identified with fairly simple in vitro techniques. Since inhibitionof aromatase activity has been demonstrated to be a common mechanism for a variety of environmentalchemicals, in vitro assays using microsomal preparations and the production of 3 H 2 O toevaluate aromatase activity provide a relatively easy and inexpensive screening method. However,chemicals that might induce aromatase activity by altering the regulation of enzyme synthesis orstability require the use of in vitro whole cell systems and/or in vivo studies that evaluate multiplearomatase-containing tissues. Finally, the design of in vivo studies is critical for understanding (1)whether a chemical has a direct effect on aromatase activity versus an indirect effect mediatedthrough a change in the regulation of the hypothalamic-pituitary-gonadal axis and (2) whether anyeffect on aromatase is tissue dependent. For example, circulating estrogen concentrations areinfluenced by the levels and cyclicity of pituitary gonadotropins. 76 Thus, the evaluation of aromataseactivity after longer exposures to a given chemical may not reflect its primary mode of action. Additionally,since aromatase expression is under the control of tissue-specific promoters regulated bydifferent cohorts of transcription factors, 77 it is important to evaluate multiple tissues for aromataseactivity in the in vivo studies. In some cases there could be local alterations in estrogen biosynthesisin a tissue that would not be reflected by an alteration of overall circulating hormone levels. 78A. Uterotrophic AssayIII. IN VIVO ASSAYS FOR THE DETECTIONOF ENDOCRINE DISRUPTING CHEMICALSThe rodent uterotrophic assay, recommended by EDSTAC as one of the core in vivo assays for theTIS, is generally accepted as a robust, technically simple, and reliable method for detectingestrogenic activity. The assay was first developed during the mid-1930s for use in both mice andrats, 79–81 and continues to be routinely used today as the gold standard to detect both potent andweak estrogen agonists 82 as well as antagonists. 83,84 Specifically, the assay measures the ability ofa test chemical to significantly increase the weight of the rodent uterus after three consecutive daysof exposure, or in the case of an antagonist, to prevent an estrogen-dependent increase in uterineweight. The assay is based upon an estrogen mode of action that includes estrogen binding to itsreceptor, initiation of gene transcription, and induction of uterine growth. 85,86 A number of studieshave correlated in vitro ER binding affinity with a uterotrophic response following in vivo exposureto a broad range of pharmaceuticals and environmental chemicals. 87–89In collaboration with the U.S. EPA, the OECD has agreed to direct the optimization andvalidation of the rodent uterotrophic assay for the TIS battery. The OECD has concentrated itsinternational validation program on evaluating the performance of two versions of the uterotrophicassay, one using sexually immature female rats and the other using adult ovariectomized (OVX)rats. Both of these versions provide an animal model that is devoid of endogenous estrogen, therebyensuring that any observed estrogenic response will be a direct influence of the test chemical.Owens and Ashby 82 have published an overview of the OECD protocol for the uterotrophic assay,along with an extensive review of the biological basis and use of the assay throughout the past 60years. In brief, sexually immature female (less than 21 days of age) or adult OVX rats (approximately60 days of age at ovariectomy with a minimum of 14 days’ recovery) are dosed with a testchemical by oral gavage or subcutaneous injection for three consecutive days, at approximately24-h intervals. Body weights are monitored daily, and the absolute uterine weights of both the wetuterus (includes imbibed fluid) and blotted uterus are recorded at necropsy, 24 h following the final© 2006 by Taylor & Francis Group, LLC