Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Aromatase inhibition leading to reproductive dysfunction||MolecularInitiatingEvent|
|Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation||MolecularInitiatingEvent|
Key Event Description
Inhibition of cytochrome P450 aromatase (CYP19; specifically cyp19a1a in fish).
Site of action: The site of action for the molecular initiating event is the ovarian granulosa cells.
While many vertebrates have a single isoform of aromatase, fish are known to have two isoforms. CYP19a1a is predominantly expressed in ovary while cyp19a1b is predominantly expressed in brain (Callard et al. 2001; Cheshenko et al. 2008). For the purposes of this MIE, when applied to fish, the assumed effect is on cyp19a1a. However, given that both isoforms show similar sensitivity to aromatase inhibitors (Hinfray et al. 2006) and catalyze the same reaction, discrimination of specific isoforms is not viewed as critical in relative to determining downstream key events resulting from aromatase inhibition in ovarian granulosa cells.
Responses at the macromolecular level: Aromatase catalyzes three sequential oxidation steps (i.e., KEGG reactions R02501, R04761, R03087 or R01840, R04759, R02351; http://www.genome.jp/kegg/pathway.html) involved in the conversion of C-19 androgens (e.g., testosterone, androstenedione) to C-18 estrogens (e.g., 17β-estradiol, estrone). Aromatase inhibitors interfere with one or more of these reactions, leading to reduced efficiency in converting C-19 androgens into C-18 estrogens. Therefore, inhibition of aromatase activity results in decreased rate of 17β-estradiol (and presumably estrone) production by the ovary.
How It Is Measured or Detected
Measurement/detection: Aromatase activity is typically measured by evaluating the production of tritiated water released upon the aromatase catalyzed conversion of radio-labeled androstenedione to estrone (Lephart and Simpson 1991). Aromatase activity can be measured in cell lines exposed in vitro (e.g., human placental JEG-3 cells and JAR choriocarcinoma cells, (Letcher et al. 1999); H295R human adrenocortical carcinoma cells (Sanderson et al. 2000)). Aromatase activity can also be quantified in tissue (i.e., ovary or brain) from vertebrates exposed in vivo (e.g., (Villeneuve et al. 2006; Ankley et al. 2002). In vitro aromatase assays are amenable to high throughput and have been included in nascent high throughput screening programs like the US EPA ToxcastTM program.
Domain of Applicability
Taxonomic applicability: Aromatase (CYP19) orthologs are known to be present among most of the vertebrate lineage, at least down to the cartilaginous fishes. Orthologs have generally not been found in invertebrates, however, CYP19 was detected in the invertebrate chordate, amphioxus and analysis of conservation of gene order and content suggests a possible origin among primitive chordates (Castro et al. 2005). Fishes generally have two aromatase isoforms, cyp19a1a which is predominantly expressed in ovary and cyp19b, predominantly expressed in brain (Callard et al. 2001). Given that cyp19a1a is dominant isoform expressed in ovary and both isoforms appear to show similar sensitivity to aromatase inhibitors (Hinfray et al., 2006), for the purpose of this key event which focuses on gonadal aromatase activty, distinction of effects on one isoform versus the other are considered negligible. Total activity, without regard to isoform can be considered.
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event
Characterization of chemical properties: Chemicals are known to inhibit aromatase activity through two primary molecular mechanisms. Steroid-like structures can inhibit the enzyme at its active site, with structures having ∆4 positioned double bonds generally acting as stronger inhibitors than those with ∆5 positioned double bonds (Petkov et al. 2009). Non-steroidal aromatase inhibitors generally act by interfering with electron transfer via the cytochrome P450 heme group of the aromatase enzyme, with greater nucleophilicity of the heteroatom contributing to greater potency as an inhibitor (Petkov et al. 2009). Petkov et al. (Petkov et al. 2009) have provided a detailed analysis of structural categorization of chemicals as potential steroidal or non-steroidal aromatase inhibitors.
- Petkov PI, Temelkov S, Villeneuve DL, Ankley GT, Mekenyan OG. 2009. Mechanism-based categorization of aromatase inhibitors: a potential discovery and screening tool. SAR QSAR Environ Res 20(7-8): 657-678.
- Lephart ED, Simpson ER. 1991. Assay of aromatase activity. Methods Enzymol 206: 477-483.
- Letcher RJ, van Holsteijn I, Drenth H-J, Norstrom RJ, Bergman A, Safe S, et al. 1999. Cytotoxicity and aromatase (CYP19) activity modulation by organochlorines in human placental JEG-3 and JAR choriocarcinoma cells. Toxico App Pharm 160: 10-20.
- Sanderson J, Seinen W, Giesy J, van den Berg M. 2000. 2-chloro-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estrogenicity. Toxicol Sci 54: 121-127.
- Villeneuve DL, Knoebl I, Kahl MD, Jensen KM, Hammermeister DE, Greene KJ, et al. 2006. Relationship between brain and ovary aromatase activity and isoform-specific aromatase mRNA expression in the fathead minnow (Pimephales promelas). Aquat Toxicol 76(3-4): 353-368.
- Ankley GT, Kahl MD, Jensen KM, Hornung MW, Korte JJ, Makynen EA, et al. 2002. Evaluation of the aromatase inhibitor fadrozole in a short-term reproduction assay with the fathead minnow (Pimephales promelas). Toxicol Sci 67: 121-130.
- Castro LF, Santos MM, Reis-Henriques MA. 2005. The genomic environment around the Aromatase gene: evolutionary insights. BMC Evol Biol 5: 43.
- Callard GV, Tchoudakova AV, Kishida M, Wood E. 2001. Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish. J Ster Biochem Mol Biol 79: 305-314.
- Cheshenko K, Pakdel F, Segner H, Kah O, Eggen RI. Interference of endocrine disrupting chemicals with aromatase CYP19 expression or activity, and consequences for reproduction of teleost fish. Gen Comp Endocrinol. 2008 Jan 1;155(1):31-62.
- Hinfray N, Porcher JM, Brion F. Inhibition of rainbow trout (Oncorhynchus mykiss) P450 aromatase activities in brain and ovarian microsomes by various environmental substances. Comp Biochem Physiol C Toxicol Pharmacol. 2006 Nov;144(3):252-62