Cardiac Abnormalities Associated With Danazol-Induced Embryopathy in Zebrafish

Danazol, and other synthetic androgens, are currently the subject of renewed interest in the clinical management of several diseases and disorders—from telomere diseases to multi-drug resistant cancer. However, very little work has studied its effect on developing organisms and it has never before been tested in Danio rerio. This project aimed to determine a non-toxic dosing program of Danazol in D. rerio and to uncover the effect of androgen exposure during embryonic development. Following treatment at 24- to 26-hours-post-fertilization, embryos show a significant decrease in basal heart rate and further evidence of cardiovascular pathology. These findings suggest that in addition to widely reported accounts of virilization in the human and rodent models, disruption to androgen physiology during early development poses increased risk for errors to normal cardiogenesis and function.

Hello, my name is Jackson Kair, and I am a senior in the Biology Department graduating at the end of the summer term. Today, I’m here to talk about my project, “Cardiac Abnormalities Associated with Danazol-Induced Embryopathy in Zebrafish” or in more lay terms, as I have taken to calling it– “Don’t Go Stopping My Heart (with a synthetic steroid)”.

This project is the culmination of my work over the past six months studying how Danazol impacts the embryonic development of zebrafish. Although most widely used in the treatment of endometriosis, adenomyosis, and fibrocystic breast disease for over 40 years—Danazol has an extensive array of biologic effects as well as diverse clinical applications in the management of countless other diseases and disorders.

My interest in Danazol, however, is due to its role as an inhibitor of adrenal steroidogenesis— the process by which steroid hormones (such as aldosterone, progesterone, testosterone, and cortisol to name a few) are synthesized by the adrenals and the gonads.

In the adult, human model: Danazol suppresses this system through the competitive inhibition of specific enzymes expressed by the adrenal glands. Likewise, once in the bloodstream, Danazol binds avidly to the sex-hormone binding globulin—displacing estradiol and testosterone. The net effect is a significant increase in the amount of testosterone in the body and a decrease in cortisol and other steroid hormones.

My question with this project was: “how do increased levels of testosterone, or the endocrine-disrupting activity of Danazol more generally, affect normal embryonic development?”. Unlike in adults, where these hormonal disruptions are actually therapeutic, I predicted that exposure to Danazol would actually induce a model of the disorders that arise to due to abnormal adrenal ontology and function.

Specifically—because Danazol is a synthetic androgen, or hormone that regulates the development and maintenance of male sex characteristics—I predicted that exposure to the drug would result in virilizing, otherwise known as masculinizing, effects: phenotypes consistent with the intersex condition congenital adrenal hyperplasia (CAH). However, given that Danazol has never before been tested in zebrafish or another live developmental model at this stage— I didn’t have any prior work to ground these preliminary assumptions in.

Over the spring semester, as I conducted pilot studies to identify a non-lethal dosing regimen of Danazol, what I saw turned my hypothesis right on its head. Instead of observing virilizing effects, or phenotypes consistent with CAH—I saw dramatic bradycardia (or a decreased basal heart rate) along with other cardiac deformities, including pericardial and yolk sack edema and blood pooling.

The goal of this project moving forward, and over the summer term, was to repeat the same drug trials, but over a statistically significant sample size—allowing me to quantitatively describe the effects of the drug treatment, specifically the reduction in heart rate.

To conduct my experiment, embryos were obtained from the zebrafish colony housed in the Animal Facility at the Krasnow Center for Advanced Study. Once acquired, any coagulated (or dead) embryos were removed and those that remained were then sorted into four petri dishes such that each contained 75 healthy embryos.

The interrenal gland, the zebrafish organ equivalent to the mammalian adrenal glands, begins to form between 24 to 30-hours-post fertilization (abbreviated to hpf)—and the steroidogenic enzymes bound by Danazol begin to be transcribed at 24 hpf. As a result, I chose to treat the embryos by exposing them to Danazol (solubilized in 5 microliters of DMSO) between 24 to 26 hpf.

Every trial consisted of a control group (egg medium with 5 microliters of DMSO) and three treatment groups (at 0.2, 0.4, and 0.6 micrograms/mL of Danazol). A starting sample size of 300 embryos were used for each trial—and I completed 3 trials over the course of the summer.

Beginning at 72 hpf, one at a time, I recorded a 15-second video of every surviving embryo in all four groups from a lateral view. I then counted the heart rate for each embryo in every video.

Here we can see the results from each of the three trials, which suggests that Danazol does not decrease heart rate in a linear, dose-dependent manner. Heart rate data was assessed using a one-way ANOVA with a post-hoc Tukey-Kramer test for multiple comparisons. This allowed me to determine if there was a statistically significant difference between the test groups, and if so, which.

For all three trials, there was found to be a statistically significant decrease in heart rate between the control group and all three treatment groups—and, with the exception of trial 1, there was no statistically significant difference in heart rate between the three treatment groups. The significant difference between the 0.2 ug/mL and 0.4 ug/mL groups in trial 1 is likely due to experimental error and not attributable to the effects of the drug.

These findings demonstrate that in addition to widely reported accounts of virilization, exposure to androgens during embryonic development poses significant risk for cardiovascular pathology. Future work in this regard may seek to identify Danazol’s mechanism of action in interfering with heart organogenesis by recording data at the 30 and 48-hpf mark to study errors in heart looping and chamber ballooning or directly measuring the levels of steroid hormones in circulation with ELISA assays.

Finally, I would like to thank my mentor Dr. Valerie Olmo—thank you for your constant support and guidance, both with this project and over the course of my senior year. I would also like to thank Dr. Karen Lee and the rest of the OSCAR team for funding my work and giving me this opportunity. And finally—I would like to thank Nathan Ridings for his filming/audio/and editing expertise. Thank you for listening.