Establishment of cytochrome P450 1a gene-knockout Javanese medaka, Oryzias javanicus, which distinguishes toxicity modes of the polycyclic aromatic hydrocarbons, pyrene and phenanthrene

Abstract

Cytochrome P450 1a (Cyp1a) is an important enzyme for metabolism of organic pollutants. To understand its reaction to polycyclic aromatic hydrocarbons (PAHs), we knocked out this gene in a marine model fish, Javanese medaka, Oryzias javanicus, using the CRISPR/Cas 9 system. A homozygous mutant (KO) strain with a four-base deletion was established using an environmental DNA (eDNA)-based genotyping technique. Subsequently, KO, heterozygous mutant (HT), and wild-type (WT) fish were exposed to model pollutants, pyrene and phenanthrene, and survivorship and swimming behavior were analyzed. Compared to WT, KO fish were more sensitive to pyrene, suggesting that Cyp1a transforms pyrene into less toxic metabolites. Conversely, WT fish were sensitive to phenanthrene, suggesting that metabolites transformed by Cyp1a are more toxic than the original compound. HT fish showed intermediate results. Thus, comparative use of KO and WT fish can distinguish modes of pollutant toxicity, providing a deeper understanding of fish catabolism of environmental pollutants.

Keywords: Acute toxicity; CRISPR/Cas 9; Metabolism; Polyaromatic hydrocarbon (PAH); cyp1a.