Abstract
Pyrrolizidine alkaloids (PAs) are common phytotoxins with both hepatotoxicity and pneumotoxicity. Hepatic cytochrome P450 enzymes are known to bioactivate PAs into reactive metabolites, which can interact with proteins to form pyrrole-protein adducts and cause intrahepatic cytotoxicity. However, the metabolic and initiation biochemical mechanisms underlying PA-induced pneumotoxicity remain unclear. To investigate the in vivo metabolism basis for PA-induced lung injury, this study used mice with conditional deletion of the cytochrome P450 reductase (Cpr) gene and resultant tissue-selective ablation of microsomal P450 enzyme activities. After oral exposure to monocrotaline (MCT), a pneumotoxic PA widely used to establish animal lung injury models, liver-specific Cpr-null (LCN) mice, but not extrahepatic Cpr-low (xh-CL) mice, had significantly lower level of pyrrole-protein adducts in the serum, liver and lungs compared with wild-type (WT) mice. While MCT-exposed LCN mice had significantly higher blood concentration of intact MCT, compared to MCT-exposed WT or xh-CL mice. Consistent with the MCT in vivo bioactivation data, MCT-induced lung injury, represented by vasculature damage, in WT and xh-CL mice but not LCN mice. Furthermore, reactive metabolites of MCT were confirmed to exist in the blood efflux from the hepatic veins of MCT-exposed rats. Our results provide the first mode-of-action evidence that hepatic P450s are essential for the bioactivation of MCT, and blood circulating reactive metabolites of MCT to the lung causes pneumotoxicity. Collectively, this study presents the scientific basis for the application of MCT in animal lung injury models, and more importantly, warrants public awareness and further investigations of lung diseases associated with exposure to not only MCT but also different PAs.
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Abbreviations
- PAs:
-
Pyrrolizidine alkaloids
- MCT:
-
Monocrotaline
- PAH:
-
Pulmonary arterial hypertension
- Dehydro-PAs:
-
Dehydro-pyrrolizidine alkaloids
- DHP:
-
( ±)-6,7-Dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine
- RBCs:
-
Red blood cells
- CPR:
-
NADPH-cytochrome P450 reductase
- LCN:
-
Liver-specific Cpr-null
- xh-CL:
-
Extrahepatic Cpr-low
- WT:
-
Wild type
- LC–MS:
-
Liquid chromatography–mass spectrometry
- GSH:
-
Glutathione
- ALT:
-
Alanine aminotransferase
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Acknowledgements
The study was supported by Research Grants Council of Hong Kong Special Administrative Region (GRF Project No. 14160817, to G. Lin), CUHK Direct Grant (Project No. 4054376, to G. Lin), and a grant from the National Institute of Environmental Health Sciences, National Institutes of Health (R01 ES020867, to X. Ding). We thank Ms. Weizhu Yang for assistance with mouse breeding and genotyping analysis, Dr. Lei Yin and Dr. Xiangmeng Wu for assistance with analytical instruments, and Dr. Weiguo Han and Dr. Nataliia Kovalchuk for assistance with histological assays.
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YH, WL, XD, and GL conceived the idea and designed the research studies. YH, WL, LD, XF, and JM conducted experiments and acquired data. YH and WL analyzed data and wrote the manuscript. PPF and QYZ revised the manuscript. XD and GL gave critical review of the manuscript.
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He, Y., Lian, W., Ding, L. et al. Lung injury induced by pyrrolizidine alkaloids depends on metabolism by hepatic cytochrome P450s and blood transport of reactive metabolites. Arch Toxicol 95, 103–116 (2021). https://doi.org/10.1007/s00204-020-02921-0
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DOI: https://doi.org/10.1007/s00204-020-02921-0