Abstract
Cyclophosphamide is a widely used anticancer and immunosuppressive prodrug that unfortunately causes severe adverse effects, including cardiotoxicity. Although the exact cardiotoxic mechanisms are not completely understood, a link between cyclophosphamide’s pharmacologically active metabolites, namely 4-hydroxycyclophosphamide and acrolein, and the toxicity observed after the administration of high doses of the prodrug is likely. Therefore, the objective of this study is to shed light on the cardiotoxic mechanisms of cyclophosphamide and its main biotransformation products, through classic and metabolomics studies. Human cardiac proliferative and differentiated AC16 cells were exposed to several concentrations of the three compounds, determining their basic cytotoxic profile and preparing the next study, using subtoxic and toxic concentrations for morphological and biochemical studies. Finally, metabolomics studies were applied to cardiac cells exposed to subtoxic concentrations of the aforementioned compounds to determine early markers of damage. The cytotoxicity, morphological and biochemical assays showed that 4-hydroxycyclophosphamide and acrolein induced marked cardiotoxicity at µM concentrations (lower than 5 µM), being significantly lower than the ones observed for cyclophosphamide (higher than 2500 μM). Acrolein led to increased levels of ATP and total glutathione on proliferative cells at 25 µM, while no meaningful changes were observed in differentiated cells. Higher levels of carbohydrates and decreased levels of fatty acids and monoacylglycerols indicated a metabolic cardiac shift after exposure to cyclophosphamide’s metabolites, as well as a compromise of precursor amino acids used in the synthesis of glutathione, seen in proliferative cells’ metabolome. Overall, differences in cytotoxic mechanisms were observed for the two different cellular states used and for the three molecules, which should be taken into consideration in the study of cyclophosphamide cardiotoxic mechanisms.
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Abbreviations
- ACRO:
-
Acrolein
- AOP:
-
Adverse outcome pathways
- ATP:
-
Adenosine 5’-triphosphate
- CYA:
-
Cyclophosphamide
- FBS:
-
Foetal bovine serum
- GC–MS:
-
Gas chromatography coupled to mass spectrometry
- GSH:
-
Reduced glutathione
- HCYA:
-
4-Hydroxycyclophosphamide
- HS-SPME:
-
Headspace solid-phase microextraction
- HS:
-
Horse serum
- MTT:
-
3-(4,5-Dimethylthiazol-2-yl) diphenyltetrazolium bromide
- MVA:
-
Multivariate analysis
- NAC:
-
N-Acetylcysteine
- NR:
-
Neutral red
- PCA:
-
Principal component analysis
- PFBHA:
-
O-(2,3,4,5,6-Pentafluorobenzyl) hydroxylamine hydrochloride
- PLS-DA:
-
Partial least squares discriminant analysis
- PTFE:
-
Polytetrafluoroethylene
- QC:
-
Quality control
- RT:
-
Retention time
- ROS:
-
Reactive oxygen species
- SD:
-
Standard deviation
- tGSH:
-
Total glutathione
- VCC:
-
Volatile carbonyl compounds
- VIP:
-
Variable importance to projection
- VOC:
-
Volatile organic compounds
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Acknowledgements
This work is financed by national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB. VMC acknowledges her grant (SFRH/BHD/110001/2015), received by Portuguese national funds through Fundação para a Ciência e Tecnologia (FCT), IP, under the Norma Transitória DL57/2016/CP1334/CT0006.
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Dionísio, F., Araújo, A.M., Duarte-Araújo, M. et al. Cardiotoxicity of cyclophosphamide’s metabolites: an in vitro metabolomics approach in AC16 human cardiomyocytes. Arch Toxicol 96, 653–671 (2022). https://doi.org/10.1007/s00204-021-03204-y
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DOI: https://doi.org/10.1007/s00204-021-03204-y