In the original article wrong unites were quoted in Table 3 (page 508) and Table 4 (page 510) as well as in the paragraph 3.2 Core chemical exposure experiments on page 509. Also in paragraph 2.3 Selection and testing of chemicals the link to the Supplemental Materials (ESM) was missing.

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The OECD validation program of the H295R steroidogenesis assay: Phase 3. Final inter-laboratory validation study

Background, goals, and scopeIn response to increasing concerns regarding the potential of chemicals to interact with the endocrine system of humans and wildlife, various national and international programs have been initiated with the aim to develop new guidelines for the screening and testing of these chemicals in vertebrates. Here, we report on the validation of an in vitro assay, the H295R steroidogenesis assay, to detect chemicals with the potential to inhibit or induce the production of the sex steroid hormones testosterone (T) and 17β-estradiol (E2) in preparation for the development of an Organization for Economic Cooperation and Development (OECD) test guideline.MethodsA previously optimized and pre-validated protocol was used to assess the potential of 28 chemicals of diverse structures and properties to validate the H295R steroidogenesis assay. These chemicals are comprised of known endocrine-active chemicals and “negative” chemicals that were not expected to have effects on the targeted endpoints, as well as a number of test chemicals with unknown modes of action at the level of the steroidogenic pathway. A total of seven laboratories from seven countries participated in this effort. In addition to effects on hormone production, confounding factors, such as cell viability and possible direct interference of test substances with antibody-based hormone detection assays, were assessed. Prior to and during the conduct of exposure experiments, each laboratory had to demonstrate that they were able to conduct the assay within the margin of predefined performance criteria.ResultsWith a few exceptions, all laboratories met the key quality performance parameters, and only 2% and 7% of all experiments for T and E2, respectively, were excluded due to exceedance of these parameters. Of the 28 chemicals analyzed, 13 and 14 tested affected production of T and E2, respectively, while 11 and 8 did not result in significant effects on T and E2 production, respectively. Four and six chemicals produced ambiguous results for effects on T and E2 production, respectively. However, four of these cases each for T and E2 were associated with only one laboratory after a personnel change occurred. Significant interference of test chemicals with some of the antibody-based hormone detection systems occurred for four chemicals. Only one of these chemicals, however, significantly affected the ability of the detection system to categorize the chemical as affecting E2 or T production.Discussion and conclusionsWith one exception, the H295R steroidogenesis assay protocol successfully identified the majority of chemicals with known and unknown modes of interaction as inducers or inhibitors of T and E2 production. Thus it can be considered a reliable screen for chemicals that can alter the production of sex steroid hormones. One of the remaining limitations associated with the H295R steroidogenesis assay protocol is the relatively small basal production of E2 and its effect on quantifying the decreased production of this hormone with regard to the identification of weak inhibitors. An initial comparison of the data produced in this study with those from in vivo studies from the literature demonstrated the potential of the H295R steroidogenesis assay to identify chemicals affecting hormone homeostasis in whole organisms. Particularly promising was the lack of any false negatives during the validation and the very low number of false positives (1 out of 28 chemicals for each T and E2).PerspectivesBased on the results obtained during this validation study and the accordingly revised test protocols, an OECD draft test guideline was developed and submitted to the OECD working group of the national coordinators of the test guidelines program (WNT) for comments in December 2009.

The OECD validation program of the H295R steroidogenesis assay. Phase 3: Final Inter-Laboratory Validation Study. Environ Sci Pol Res

Elevated expression of P450c17 (CYP17) during testicular formation in the frog.

Immunohistochemical detection and biological activities of CYP17 (P450c17) in the indifferent gonad of the frog Rana rugosa.

Seasonal changes in testicular steroidogenesis in the toad Bufo arenarum H.

In Bufo arenarum, the biosynthesis of testosterone and 5α-dihydrotestosterone takes place through a complete 5-ene pathway, 5-androsten-3β,17β-diol being the immediate precursor of testosterone. Besides androgens, testes are able to synthesise 5α-pregnan-3,20-dione and several 3α and 20α reduced derivatives. During the breeding season, steroid biosynthesis turns from androgen to C21-steroid production. As a consequence, the cytochrome P450 17-hydroxylase, C17,20-lyase (CypP450c17) could be a key enzyme in that metabolic shift. The present study demonstrates that in testes of B. arenarum, CypP450c17 co-localises with glucose-6-phosphatase in the microsomal fraction. CypP450c17 possesses more affinity for pregnenolone than for progesterone in both non-reproductive (Km=43.76±4.63 nM and 2,170±630 nM, respectively) and reproductive (Km=37.46±4.19 nM and 3,060±190 nM, respectively) seasons. These results could explain the predominance of the 5-ene pathway for testosterone biosynthesis. Toad CypP450c17 activity is higher in the non-reproductive period than the reproductive period, suggesting that this enzyme is an important factor in toad steroidogenic changes. Animals in reproductive conditions showed a significant reduction in circulating androgens. This is in agreement with the decrease in Vmax of cytochrome P450 17-hydroxylase activity, enhancing the physiological relevance of these in vitro results.

Seasonal changes in the activity of cytochrome P450(C17) from the testis of Bufo arenarum.

Testis fragments from Bufo arenarum were incubated with [7(n)-3H]pregnenolone (P5), [1,2-3H]dehydroepiandrosterone (DHEA) and [1,2,6,7-3H]testosterone (T), and different steroid-biosynthesis inhibitors. The inhibitors used were: cyanoketone (CNK), spironolactone (SPNL) and finasteride (FIN). CNK significantly increased the recovery of 3β-hydroxy-5-ene steroids while SPNL reduced the metabolism of P5 and the production of C19-steroids. The metabolism of C19-substrates was only modified by CNK, which reduced the transformation of DHEA without modifying the metabolism of T. To determine the degree of inhibition exerted by the inhibitors used, the activities of the enzymes were estimated as the percentage of their contribution to the total steroid metabolism. CNK strongly inhibited the activity of hydroxysteroid dehydrogenase/isomerase if its contribution was estimated using both P5 and DHEA. If the analysis was made considering both activities associated to cytochrome P450 17α-hydroxylase, C17–20 lyase (P450c17), it became evident that SPNL inhibited both of them. The percent contribution of 17β-hydroxysteroid dehydrogenase (17βHSD) activity diminished in the presence of CNK only if it was estimated considering P5 and DHEA metabolism. SPNL produced a significant inhibition of 17βHSD when its contribution was estimated considering P5 metabolism. However, SPNL was insufficient if DHEA or T were considered. The effect of SPNL on the contribution of 17βHSD could be due to the reduction of C19-substrates. The activity of 5α-reductase was inhibited by CNK only if results from P5 and DHEA were considered.

N R Ceballos

Papers

Seasonal changes in the activity of cytochrome P450(C17) from the testis of Bufo arenarum.

Effects of different steroid-biosynthesis inhibitors on the testicular steroidogenesis of the toad Bufo arenarum.