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C:\Myfiles\Linda's Work\Houchens\WA 2-6 1 2 This page intentionally left blank. 3 4 Draft Steroidogenesis DRP 110 May 2002 1 6.0 DEVELOPMENTAL STATUS OF THE ASSAY AND RECOMMENDATIONS 2 FOR PREVALIDATION STUDIES 3 4 6.1 Current Status 5 6 The endpoint included in the sectioned testes assay has been evaluated in other studies. 7 However, the protocol itself has not been validated. Pending a final decision on the study 8 design, the protocol would be ready to enter the prevalidation phase. 9 10 6.2 Recommendation for Optimization of the Sectioned Testis Assay Protocol 11 12 6.2.1 Testicular Preparation Issues 13 14 Optimization of the assay could be determined for the amount of testis actually needed to 15 obtain a given level of sensitivity. For example, a single testis from an adult SD rat weighs 16 approximately 1 g. If such a testis were quarter sectioned, then each section would weigh 17 approximately 250 mg, which is the weight of the sample generally described by investigators 18 who have used quartered sections of testis. However, no documentation was found that 19 demonstrates whether smaller sections would give similar results. Thus, it would be 20 advantageous to conduct a study that investigates whether the sensitivity of the preparation is 21 affected by the amount of testicular tissue used and, if so, if there is an optimal and/or threshold 22 amount to use. The weight of the sections to be tested could range from the customary amount 23 used, i.e., 250 mg, down to an amount of tissue that represents a practical minimum, e.g., 5 to 24 10 mg. 25 26 It would also be useful to explore storage and viability of the testis and/or sections. One 27 possible scenario to assist in meeting an ICCVAM and U.S. EPA objective to reduce, refine, and 28 replace animal usage would be to use testes from animals in a separate study, that requires 29 castration in the experimental design, e.g., Hershberger studies. It may be possible to store testes 30 after removal in such a way that they remain viable, can be shipped to various locations, and are 31 used at a later date for the in vitro sectioned testis steroidogenesis assay. A storage condition, 32 stability, and viability study could be designed and tested. 33 34 6.2.2 Endpoint Issues 35 36 The importance of measuring progesterone could be evaluated. For example, it is 37 possible that the stoichiometric molar relationship for progesterone and testosterone is not 1:1. 38 If such a relationship exists and a substance inhibits progesterone production but the “pool” of 39 progesterone is sufficiently large such that the production of testosterone is not affected, then the 40 assay would not detect an effect on steroidogenesis if only testosterone were measured. 41 However, this effect would be observed if progesterone were measured. This possibility could 42 be determined during the initial experiments used to optimize the assay. 43 Draft Steroidogenesis DRP 111 May 2002 1 The stability of the media samples could be determined during these initial studies. Since 2 the assay lends itself to multiple endpoints but the assay is most efficient by measuring a single 3 important endpoint, i.e., testosterone, it would be useful to evaluate the length of time that the 4 media could be stored and used at a later date to measure other endpoints, e.g., progesterone, 5 estradiol. This information could easily be obtained by conducting storage stability studies of 6 the media collected from studies used to optimize the experimental design. 7 8 Another experimental design factor that could be optimized by experimental 9 determination is the number of collection time points. The current study design includes four 10 time points. Media samples are collected at 1, 2, 3, and 4 hours after the incubation is initiated. 11 The possibility exists that other time points are better suited to characterize the effect of a 12 substance on steroid hormone production. Along those same lines, perhaps fewer time points are 13 equally as useful to measure an effect. Statistical analysis could be used to determine whether 14 concentrations measured at 1 and 4 hours provide no more or no less information than that 15 obtained by measuring samples at four different time points. Such information could be used to 16 reduce extraneous collection and analysis steps. 17 18 6.2.3 Stimulation Factor Issues 19 20 The initial studies need to optimize the concentration of stimulant added to the testicular 21 preparation. The stimulant planned for use is hCG. The amount of stimulant used is important 22 because it can affect whether a steady proportional increase in steroid hormone production 23 occurs over the entire duration of the incubation period. Also, a measure of the variability of 24 different lots of hCG could be determined during such experiments. This would serve to provide 25 needed information about factors that affect the variability of the assay. 26 27 6.3 Recommendation for Sectioned Testis Assay Prevalidation Studies 28 29 Prevalidation studies following the ICCVAM validation process should be initiated. 30 Prevalidation studies should include evaluation of six to eight substances to establish the 31 database for the validation studies. It is recommended that the study be performed using test 32 substances with different chemical classifications, as well as varying sites and/or mechanisms of 33 action, which will aid in the development of the prevalidation database for the assay. The 34 recommended positive and negative control test substances were selected based on their sites of 35 action, i.e., aminoglutethimide inhibits P450SCC and finasteride inhibits 5α-hydroxylase, which is 36 not found in the testes. Other test substances of interest that are recommended for testing in the 37 prevalidation studies include: 38 39 ! bisphenol A (inhibits steroidogenic signal transduction) 40 ! lindane (inhibits signal transduction and the StAR protein) 41 ! ketoconazole (a weak imidazole anti-fungal; inhibits P450SCC and aromatase) 42 ! genistein (a weak phytoestrogen/flavonoid; inhibits 3β-HSD) Draft Steroidogenesis DRP 112 May 2002 1 ! flutamide (inhibits P450c17) 2 ! econazole (a potent imidazole; inhibits aromatase). 3 4 6.4 Recommendation for Further Development of Cell Line Methods 5 6 In addition to the prevalidation studies for the in vitro sectioned testis assay, further 7 characterization and development of the cell lines as screening tool assays is recommended. 8 Based on the information summarized in Section 4.5 (Table 4-10), there are 2 to 3 cell lines that 9 could be studied further for their possible use as assays for testing substances for steroidogenesis 10 altering activity. The recommended cell lines are the MA-10, R2C, and H295R cells. These cell 11 lines are recommended because they represent cell lines from three different species, i.e., mouse, 12 rat, and human, respectively. The Leydig-like steroidogenic properties of the MA-10 cell line 13 have been characterized to the greatest extent and exhibit many of the properties of Leydig cells 14 up through the production of progesterone. In addition, the MA-10 cell line will provide a good 15 standard for comparison as the properties of the other cell lines are more fully investigated, 16 which is also recommended in the prevalidation studies. 17 18 Another reason that these cell lines are recommended for further study is that they are 19 readily available; Dr. M Ascoli (University of Iowa, Ames, Iowa) holds the MA-10 cell line and 20 the American Type Culture Collection (ATCC) stocks the R2C and H295R cell lines. As for the 21 H295R cell line, it is unique in that it is an immortalized human cell line and, although it is 22 derived from non-gonadal tissue, it appears to possess many of the properties that would make it 23 a viable tool for testing substances for their effects on both the gonadal and adrenal steroidogenic 24 pathways. Finally, the goal of the EPA to develope non-animal assays could be further attained 25 if one or more of these cell lines were found to be useful investigative paradigms. For these 26 reasons, it is recommended that consideration be given to further study of cell lines as screening 27 tools for identifying substances with steroidogenic altering activity. 28 29 30 Draft Steroidogenesis DRP 113 May 2002 1 2 This page intentionally left blank. 3 4 Draft Steroidogenesis DRP 114 May 2002 1 7.0 REFERENCES 2 3 Abayasekara, D.R.E., Kurlak, L. O., Band, A. M., Sullivan, M.H.F., and Cooke, B. A. (1991). 4 Effect of cell purity, cell concentration, and incubation conditions on rat testis Leydig cell 5 steroidogenesis. In Vitro Cell. Dev. Biol. 27A, 253–259. 6 7 Adebanjo, O. A., Igietseme, J., Huang, C. L., and Zaidi, M. (1998). The effect of extracellularly 8 applied divalent cations on cytosolic Ca2+ in murine Leydig cells: evidence for a Ca2+-sensing 9 receptor. J. Physiol., 513 (Pt 2):399-410. 10 11 Allen, E. and Doisy, E. (1924). The induction of a sexually mature condition in immature 12 females by injection of the ovarian follicular hormone. Am. J. Physiol. 69, 577-588. 13 14 Andersen, H. R., Vinggaard, A. M., Rasmussen, T. H., Gjermandsen, I. M., and 15 Bonefeld-Jorgensen, E. C. (2002). Effects of currently used pesticides in assays for estrogencity, 16 androgencity, and aromatase activity in vitro. Tox. Appl. Pharmacol., 179:1-12. 17 18 Anderson, S. A., Sauls, H. R., Pearce, S. W., and Fail, P. A. (1992). Endocrine responses after 19 boric acid exposure for 2, 9, and 14 days in cannulated male CD rats.
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