New toxicogenetic insights and ranking of the selected pharmaceuticals belong to the three different classes: A toxicity estimation to confirmation approach.

Tetracycline hydrochloride (TH), indomethacin (IM), and bezafibrate (BF) belong to the three different important classes of pharmaceuticals, which are well known for their toxicity and environmental concerns. However, studies are still elusive to highlight the mechanistic toxicity of these pharmaceuticals and rank them using both, the toxicity prediction and confirmation approaches. Therefore, we employed the next generation toxicity testing in 21st century (TOX21) tools and estimated the in vitro/vivo toxic endpoints of mentioned pharmaceuticals, and then confirmed them using in vitro/vivo assays. We found significant resemblance in the results obtained via both approaches, especially in terms of in vivo LC50 s and developmental toxicity that ranked IM as most toxic among the studied pharmaceuticals. However, TH appeared most toxic with the lowest estimated AC50s, the highest experimental IC50s, and DNA damages in vitro. Contrarily, IM was found as congener with priority concern to activate the Pi3k-Akt-mTOR pathway in vitro at concentrations substantially lower than that of TH and BF. Further, IM exposure at lower doses (2.79-13.97 μM) depressed the pharmaceuticals detoxification phase I (CYP450 s), phase II (UGTs, SULTs), and phase III (TPs) pathways in zebrafish, whereas, at relatively higher doses, TH (2.08-33.27 μM) and BF (55.28-884.41 μM) partially activated these pathways, which ultimately caused the developmental toxicity in the following order: IM > TH > BF. In addition, we also ranked these pharmaceuticals in terms of their particular toxicity to myogenesis, hematopoiesis, and hepatogenesis in zebrafish embryos. Our results revealed that IM significantly affected myogenesis, hematopoiesis, and hepatogenesis, while TH and BF induced prominent effects on hematopoiesis via significant downregulation of associated genetic markers, such as drl, mpx, and gata2a. Overall, our findings confirmed that IM has higher toxicity than that of TH and BF, therefore, the consumption of these pharmaceuticals should be regulated in the same manner to ensure human and environmental safety.