The continuous accumulation of senescent hematopoietic stem progenitors (HSPCs) contributes to hematopoietic damage. Benzene is a confirmed human carcinogen, and its damage to HSPCs is a key event in benzene poisoning. However, whether the environmental dose of benzene is involved in HSPC damage by inducing cellular senescence has not been reported. Here, male C57BL/6 J mice were exposed to benzene vapour for 12 weeks (0, 10, 50 ppm), and mouse hematopoietic progenitor cell line FDC-P1 was exposed to benzene metabolite 1,4-BQ for 24 h (0, 5, 10 μM). In vivo and in vitro models combined with single-cell RNA sequencing have shown that benzene and its metabolites caused senescence in whole bone marrow and hematopoietic progenitor cells. Proteomics showed that benzene exposure significantly up-regulated interferon regulatory factor 4 (Irf4) in the bone marrow. Irf4 inhibition alleviated cellular senescence and hematopoietic damage, suggesting that Irf4 is a key molecule in benzene-induced hematopoietic cell senescence. Mechanistically, benzene caused a decrease in branched-chain amino acids (BCAAs) in whole bone marrow, hematopoietic progenitor cells, and plasma, and an increase in the BCAA catabolic enzyme Bcat1, mitochondrial ROS, and Bckdh activity. Irf4 inhibition down-regulated Bcat1, alleviated mitochondrial oxidative stress-dependent Bckdh abnormality, and up-regulated BCAAs. BCAA supplementation effectively alleviated benzene-induced cellular senescence and hematopoietic damage. In conclusion, the study identified that Irf4 triggered benzene-induced hematopoietic progenitor cell senescence and hematopoietic damage by mitochondrial injury-induced excessive BCAA catabolism. This study provides new ideas on the molecular mechanism of benzene-induced hematopoietic damage from the perspective of metabolism and senescence.
The dysregulated levels of branched chain amino acids (BCAA) contribute to renal fibrosis in chronic kidney disease (CKD), yet specific analysis of BCAA contents and how they are regulated still remain unclear. It is therefore of great scientific interest to understand BCAA catabolism in CKD and develop a sensitive method for simultaneous determination of individual BCAA and their metabolites branched chain α-ketoacids (BCKA). In this work, the important role of BCAA metabolism that drives renal fibrosis in the process of CKD was first revealed by using transcriptomics. The key target genes controlling BCAA metabolism were then validated, that is, mRNA levels of BCKDHA and BCKDHB , the regulating rate-limiting enzymes during BCAA metabolism were abnormally reduced by quantitative PCR (qPCR), and a similar drop-off trend of protein expression of BCKDH, HIBCH and MCCC2 that are closely related to BCAA metabolism was also confirmed by western blotting. Furthermore, we established a novel strategy that simultaneously determines 6 individual BCAA and BCKA in serum and tissue. The method based on dansylhydrazine derivatization and ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC-QQQ-MS) achieved to simultaneously determine the contents of BCAA and BCKA, which is efficient and stable. Compared with normal rats, levels of BCAA including leucine, isoleucine and valine in serum and kidney of CKD rats was decreased, while BCKA including α-ketoisocaproic acid, α-ketomethylvaleric acid and α-ketoisovaleric acid was increased. Together, these findings revealed the abnormality of BCAA metabolism in driving the course of kidney fibrosis and CKD. Our current study sheds new light on changes in BCAA metabolism during CKD, and may facilitate development of drugs to treat CKD and renal fibrosis.
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