Mechanistic Evaluation of Benzo[a]pyrene's Developmental Toxicities Mediated by Reduced Cyp19a1b Activity

Benzo[a]pyrene (BaP) is a ubiquitous environmental contaminant that is both an endocrine disruptor and a carcinogen. Aromatase (CYP19) is a key enzyme in steroidogenesis that is responsible for conversion of androgens to estrogens and thus plays a key role in steroid homeostasis. We hypothesized that some of the adverse outcomes of early developmental exposure to BaP are the result of reduced Cyp19a1b activity. Our goal was to investigate the role of estrogen homeostasis during early development and determine the role of aromatase inhibition as a relevant mechanism in BaP's developmental toxicities. One-cell zebrafish embryos were injected with a Cyp19a1b-morpholino (MO) or control-MO. Other non-injected embryos were exposed to waterborne BaP, fadrozole (a Cyp19 inhibitor), estradiol (E2), BaP + E2, Cyp19a1b MO + E2, or fadrozole + E2 for 96 hours post-fertilization (hpf). Adverse outcomes were compared between treatments, and the ability of E2 co-exposure to rescue each observed dysmorphology was assessed. BaP significantly decreased cyp19a1b gene expression in 96 hpf zebrafish larvae homogenates. Concentrations of E2 in 48 hpf larvae were significantly decreased by BaP, fadrozole and Cyp19a1b-MO. Cumulative mortality of zebrafish larvae was significantly increased following BaP or fadrozole exposure or Cyp19a1b knockdown compared to controls. E2 co-treatment rescued mortality caused by 10 μg/L BaP, 10 μg/L fadrozole, or Cyp19a1b-MO. In a treatment-blinded morphological assessment of larvae at 96 hpf, several phenotypes were negatively impacted by BaP, fadrozole, or Cyp19a1b knockdown and rescued by exogenous E2 co-treatment; these included body length, optic vesicle size, swim bladder inflation, pericardial and abdominal edema, and incidence of normal larval tail shape. Abnormal pectoral fins were caused by BaP exposure only. Uninflated swim bladders were caused by all treatments including E2 alone. Our results indicate that certain BaP-mediated adverse developmental outcomes were mechanistically in accordance with BaP-mediated Cyp19a1b inhibition.