In silico flux balance analysis of diclofenac-induced gonadal toxicity in zebrafish (Danio rerio).

Non-steroidal anti-inflammatory drugs (NSAIDs) are cyclooxygenase (COX) inhibitors that can cause wide-ranging metabolic disruptions. We used the in silico systems biology framework of flux balance analysis (FBA) to study the effects of diclofenac (DICL) on the gonadal metabolic physiology of adult male and female zebrafish (Danio rerio). We exposed zebrafish to 1 µM DICL for 4 days, then processed their gonads for quantitative PCR or RNA-sequencing analysis. We used up- and down-regulated gene expression changes to re-constrain the min/max catalytic bounds for reactions in a zebrafish stoichiometric metabolism model. FBA simulations recapitulated COX inhibition and indicated a shunting of arachidonic acid (ARA) flux through ancillary lipoxygenase and CYP450 ω-hydroxylase and/or epoxygenase pathways. FBA also predicted dysregulation of bile acid synthesis, likely due to reduced prostaglandin production (a consequence of COX inhibition). A key finding was female-specific downregulation of keratan sulfate (KS) metabolism, implicating reduced viability of the vitelline envelope surrounding oocytes. Finally, FBA assessed the effects of NSAID mixtures by knocking out vulnerable genes (identified by other authors). FBA showed a dose-response relationship between increasing NSAID concentrations (< 0.1 to > 100 µM) and worsening metabolic effects, resulting in a mortality phenotype (cessation of biomass production) at the highest exposure level (> 100 µM). Our study demonstrates the versatility of the zebrafish metabolic model and FBA to assess single and/or mixture NSAID effects, and it identified a novel biomarker (KS) that may reflect compromised egg viability in spawning females.