Cyclophosphamide is used against lymphomas, solid tumors, namely breast, ovarian, bone and soft tissue tumors, in bone marrow transplant conditioning regimens and also in the treatment of autoimmune diseases. Despite its broad use, the application of cyclophosphamide is dose limited by its cardiotoxic effects, which have been linked to its intricate bioactivation process. In this study, we evaluated the cytotoxicity of cyclophosphamide (100 to 10000 µM) and two of its main metabolites, 4-hydroxycyclophosphamide (1 to 25 µM) and acrolein (5 to 100 µM) in AC16 cells, a human cardiomyocyte cell line. Two distinctive cellular states of AC16 cells were used: differentiated with DMEM/F12 medium + 2% horse serum and proliferative, using DMEM/F12 medium + 12.5% fetal bovine serum. The tetrazolium-based colorimetric (MTT) and the neutral red (NR) uptake assays were performed at different time-points (24, 48 or 72h) for both cell states and experimental conditions. Furthermore, metabolomic evaluation was conducted in proliferative and differentiated cells after their incubation for 24h with subtoxic concentrations LC₀₅ of cyclophosphamide, 4-hydroxycyclophosphamide and acrolein.

In AC16 proliferative cells, cyclophosphamide caused toxicity already at 2 500 µM in the MTT reduction assay after a 72-h incubation, while in the NR assay significant toxicity was only seen at 5 000 µM. For the same time-point, both tests revealed that the differentiated state showed significant toxicity only at 7 500 µM. For 4-hydroxycylophoshamide, the 1 µM concentration caused a marked cytotoxic effect in the MTT assay at 72h in the proliferative cellular state. Meanwhile, in the differentiated cells, a clear cytotoxic effect was only seen at 5 µM of 4-hydroxycylophoshamide for both tests at 48h. On the other hand, in acrolein-exposed cells, the differentiated cells were more sensitive as a concentration of 15 µM caused a cytotoxic effect in the MTT assay at 24h, in comparison to 25 µM for the proliferative state. Additionally, metabolomic profiling was performed in proliferative and differentiated cells after their incubation for 24h with subtoxic concentrations LC₀₅ of cyclophosphamide (2.5 mM for both cellular states) and 4-hydroxycyclophosphamide (5 and 1 µM for the proliferative and the differentiated cells, respectively). Since acrolein is formed from the spontaneous degradation of the 4-hydroxycyclophosphamide in a straight pathway, the concentrations of acrolein used in this study were the same as the LC₀₅ of the aforementioned metabolite and not the LC₀₅ itself of acrolein. The intracellular metabolome of the cells was examined using gas chromatography/mass spectrometry (GC/MS). A multivariate analysis of the intracellular data showed a distinctive metabolic pattern between the control and the metabolites in proliferative cells, while no changes were seen in cells in differentiated state.

With the results obtained so far, we can conclude that cyclophosphamide per se only exerts cardiotoxic effect at high concentrations, while its metabolites were cytotoxic at clinically relevant concentrations. Moreover, µM concentrations of the metabolites caused changes in metabolic pathways in AC16 cells that are being further analyzed.

AMA and VMC acknowledge FCT for their grants (SFRH/BD/107708/2015 and SFRH/BPD/110001/2015). VMC’s grant is funded by national funds through FCT – Fundação para a Ciência e a Tecnologia, I.P., under the Norma Transitória – DL57/2016/CP1334/CT0006. This work was supported by FEDER funds [Operational Program for Competitiveness Factors – COMPETE and by FCT within the project “PTDC/DTP-FTO/1489/2014 – POCI-01-0145-FEDER-016537”].