Nicotine metabolites generate craniofacial defects that are worsened by ethanol exposure

Nicotine and alcohol are two of the most consumed substances among adolescents and adults. There is great concern regarding the deleterious effects of exposure to these substances during pregnancy. Complex interactions between genes and the environment are thought to influence the severity of birth defects and can occur by altering the metabolism of substances. In humans, nicotine is mainly metabolized by CYP2A6 and variants within this enzyme associate with nicotine dependence and metabolism. Previously, we showed that embryonic nicotine exposure reduced the size of craniofacial cartilages in zebrafish. Here, we show that zebrafish metabolize nicotine similarly to humans. We demonstrate that it is not nicotine itself, but nicotine metabolites, particularly 4HPBA followed by nicotine-N'-oxide (NOX), that disrupt craniofacial development. We found that cyp2y3 mutants (the zebrafish homolog of CYP2A6) have reduced levels of nicotine metabolites and are partially protected against the craniofacial defects produced by nicotine. We also examined the effect of nicotine and ethanol co-exposure in zebrafish embryos. Surprisingly, while embryos co-exposed to nicotine and alcohol had reduced craniofacial elements, they were significantly less affected than those exposed to nicotine alone. We found that nicotine metabolites levels were decreased in embryos coexposed to nicotine and ethanol. However, alcohol worsened the craniofacial defects in embryos exposed to metabolites. Our results collectively show that nicotine is metabolized into teratogenic substances and that multifactorial interactions can alter this metabolism and subsequent teratogenicity.