The Main Metabolites of Fluorouracil + Adriamycin + Cyclophosphamide (FAC) Are Not Major Contributors to FAC Toxicity in H9c2 Cardiac Differentiated Cells

Abstract

In the clinical practice, the combination of 5-fluorouracil (5-FU) + Adriamycin (also known as doxorubicin, DOX) + cyclophosphamide (CYA) (known as FAC) is used to treat breast cancer. The FAC therapy, however, carries some serious risks, namely potential cardiotoxic effects, although the mechanisms are still unclear. In the present study, the role of the main metabolites regarding FAC-induced cardiotoxicity was assessed at clinical relevant concentrations. Seven-day differentiated H9c2 cells were exposed for 48 h to the main metabolites of FAC, namely the metabolite of 5-FU, α-fluoro-β-alanine (FBAL, 50 or 100 μM), of DOX, doxorubicinol (DOXOL, 0.2 or 1 μM), and of CYA, acrolein (ACRO, 1 or 10 μM), as well as to their combination. The parent drugs (5-FU 50 μM, DOX 1 μM, and CYA 50 μM) were also tested isolated or in combination with the metabolites. Putative cytotoxicity was evaluated through phase contrast microscopy, Hoechst staining, membrane mitochondrial potential, and by two cytotoxicity assays: the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and the neutral red (NR) lysosomal incorporation. The metabolite DOXOL was more toxic than FBAL and ACRO in the MTT and NR assays. When in combination, neither FBAL nor ACRO increased DOXOL-induced cytotoxicity. No nuclear condensation was observed for any of the tested combinations; however, a significant mitochondrial potential depolarization after FBAL 100 μM + DOXOL 1 μM + ACRO 10 μM or FBAL 100 μM + DOXOL 1 μM exposure was seen at 48 h. When tested alone DOX 1 μM was more cytotoxic than all the parent drugs and metabolites in both the cytotoxicity assays performed. These results demonstrated that DOXOL was the most toxic of all the metabolites tested; nonetheless, the metabolites do not seem to be the major contributors to FAC-induced cardiotoxicity in this cardiac model.