Identification of candidate reference chemicals for in vitro steroidogenesis assays

Abstract

The Endocrine Disruptor Screening Program (EDSP) is transitioning from traditional testing methods to integrating ToxCast/Tox21 in vitro high-throughput screening assays for identifying chemicals with endocrine bioactivity. The ToxCast high-throughput H295R steroidogenesis assay may potentially replace the low-throughput assays currently used in the EDSP Tier 1 battery to detect chemicals that alter the synthesis of androgens and estrogens. Herein, we describe an approach for identifying in vitro candidate reference chemicals that affect the production of androgens and estrogens in models of steroidogenesis. Candidate reference chemicals were identified from a review of H295R and gonad-derived in vitro assays used in methods validation and published in the scientific literature. A total of 29 chemicals affecting androgen and estrogen levels satisfied all criteria for positive reference chemicals, while an additional set of 21 and 15 chemicals partially fulfilled criteria for positive reference chemicals for androgens and estrogens, respectively. The identified chemicals included pesticides, pharmaceuticals, industrial and naturally-occurring chemicals with the capability to increase or decrease the levels of the sex hormones in vitro. Additionally, 14 and 15 compounds were identified as potential negative reference chemicals for effects on androgens and estrogens, respectively. These candidate reference chemicals will be informative for performance-based validation of in vitro steroidogenesis models.

Keywords: Androgens; Estrogens; Gonadal models; H295R; Sex hormones; Steroidogenesis.

Figure 1.

Representation of the literature search scheme used to identify candidate reference chemicals for in vitro steroidogenesis assays. A two-step systematic literature was performed to identify chemicals affecting the synthesis of androgens and estrogens. The first search was designed to identify chemicals altering the synthesis of androgens (Testosterone [T] and/or androstenedione [A4]) and/or estrogens (17β-estradiol [E2] and/or estrone [E1]) in H295R cells. Chemicals with H295R data were then used in a second literature search targeting testicular and ovarian models. RC = Reference chemical

Figure 2.. Results of the effect of 70 chemicals identified in H295R studies on T and E2 levels.

Chemicals are listed along the x-axis and the log₁₀ transformed concentrations along the y-axis. Green and red dots represent LELs (or AC50s) of studies reporting decreases and increases, respectively, in T (Panel A) or E2 (Panel B). Gray dots indicate HCT for negative results. The size of the dots is proportional to the number of studies (range 1 – 7). Plot includes OECD H295R validation results and H295R literature studies of chemicals tested in one or more concentrations. Similar plots for chemical effects on A4 and E1 are provided in supplemental figures 1 and 2, respectively. (LEL = Lowest effect level; HCT = highest concentration tested)

Figure 3.. Gonadal models represented in the literature search.

The number of studies indicating the distribution of gonadal models per animal class is represented in A. Ovarian cells include granulosa cells, theca cells, co-cultures and ovarian follicles. Primary testicular cells are represented mostly by purified Leydig cells. Leydig cell lines constitute tumor-derived cells (BLTK-1, R2C, MA-10, MLTC-1) and Leydig cells isolated from normal immature testis (TM-3). Ovary and testis explants constitute whole tissue cultures, fragments and slices. The distribution of gonadal models per mammalian animal sources are indicated in B. “Other” refers to studies using bank vole, hamster, buffalo, monkey, dog, sheep, and rabbit gonadal models.

Figure 4.. Results of the effect of 50 chemicals identified in gonadal studies on T and E2 levels.

Chemicals are listed along the x-axis and the log₁₀ transformed concentrations along the y-axis. Blue and orange dots represent LELs (or AC₅₀s) of studies reporting decreases and increases, respectively, in T (Panel A) or E2 (Panel B). Gray dots indicate HCT for negative results. The size of the dots is proportional to the number of studies (T range: 1 – 18; E2 range: 1 – 13). The plot represents literature studies testing the effect of a chemical in one or more concentrations in ovarian or testicular-derived models. (LEL = Lowest effect level; HCT = highest concentration tested).

Figure 5.. Potency values for candidate reference chemicals affecting T and E2 levels.

The log LELs (or log AC₅₀s) are indicated for positive Group 1 and Group 2 candidate reference chemicals, and the HCT is indicated for candidate negative reference chemicals. A. Plot of candidate reference chemicals affecting T levels. A total of 359 positive and 42 negative studies are plotted. The log LEL and log AC₅₀ are represented for 336 and 23 studies, respectively. Details on studies used for potency characterization is in Supplemental file 7. B. Candidate reference chemicals affecting E2 levels. A total of 322 positive and 45 negative studies are plotted. The log LEL and log AC₅₀ are represented for 302 and 20 studies, respectively. Details on studies used for potency characterization is in Supplemental file 10. Only studies testing the effect of a chemical in at least two concentrations are plotted and included for calculation of median log potency values. Green and blue dots indicate, respectively, H295R and gonadal studies showing a decrease (inhibition) on the levels of hormones; red and yellow dots represent H295R and gonadal studies indicating an increase (induction) on hormone levels. Black asterisk indicates the median log potency value. Dark and light gray dots indicate HCTs for negative chemicals. The size of the dots is proportional to the number of studies. (LEL = Lowest effect level; HCT = highest concentration tested).

Figure 6.. Candidate reference chemicals affecting both the levels of androgens and estrogens.

A. Venn diagram indicating number of group 1 and group 2 reference chemicals commonly affecting the levels of androgens and estrogens. B. Median potency values of candidate reference chemicals for T and E2. The colors designate the predominant direction of effect in the positive studies. Green and red indicate, respectively, that chemicals caused a decrease (inhibition) and increase (induction) on the levels of the sex hormones in at least 75% studies. Absence of color indicates no predominant effect. C. Venn overlap of negative chemicals for both androgens and estrogens.