Abstract
The influence of ethanol on the small molecule metabolome and the role of CYP2E1 in ethanol-induced hepatotoxicity were investigated using liquid chromatography-mass spectrometry (LC-MS)-based metabolomics platform and Cyp2e1-null mouse model. Histological and biochemical examinations of ethanol-exposed mice indicated that the Cyp2e1-null mice were more resistant to ethanol-induced hepatic steatosis and transaminase leakage than the wild-type mice, suggesting CYP2E1 contributes to ethanol-induced toxicity. Metabolomic analysis of urinary metabolites revealed time- and dose-dependent changes in the chemical composition of urine. Along with ethyl glucuronide and ethyl sulfate, N-acetyltaurine (NAT) was identified as a urinary metabolite that is highly responsive to ethanol exposure and is correlated with the presence of CYP2E1. Subsequent stable isotope labeling analysis using deuterated ethanol determined that NAT is a novel metabolite of ethanol. Among three possible substrates of NAT biosynthesis (taurine, acetyl-CoA, and acetate), the level of taurine was significantly reduced, whereas the levels of acetyl-CoA and acetate were dramatically increased after ethanol exposure. In vitro incubation assays suggested that acetate is the main precursor of NAT, which was further confirmed by the stable isotope labeling analysis using deuterated acetate. The incubations of tissues and cellular fractions with taurine and acetate indicated that the kidney has the highest NAT synthase activity among the tested organs, whereas the cytosol is the main site of NAT biosynthesis inside the cell. Overall, the combination of biochemical and metabolomic analysis revealed NAT is a novel metabolite of ethanol and a potential biomarker of hyperacetatemia.
Highlights
Ethanol-related metabolic activities induce the changes in the small molecule metabolome
After a 21-day ethanol feeding, cytochrome P450 2E1 (CYP2E1) protein was dramatically increased in the wild-type mice but remained absent in the Cyp2e1-null mice (Fig. 1B), confirming that CYP2E1 expression is responsive to ethanol treatment [25]
The results showed that the development of both microvesicular and macrovesicular steatosis around the central vein was delayed in the Cyp2e1-null mice compared with the wild-type mice (Fig. 1C)
Summary
Ethanol-related metabolic activities induce the changes in the small molecule metabolome. Results: Metabolomic analysis revealed that the level of N-acetyltaurine (NAT) in urine increases dramatically after ethanol consumption. Conclusion: Ethanol-induced NAT biosynthesis is mainly caused by a novel reaction between taurine and excessive acetate produced by ethanol metabolism. Metabolomic analysis of urinary metabolites revealed time- and dose-dependent changes in the chemical composition of urine. Subsequent stable isotope labeling analysis using deuterated ethanol determined that NAT is a novel metabolite of ethanol. In vitro incubation assays suggested that acetate is the main precursor of NAT, which was further confirmed by the stable isotope labeling analysis using deuterated acetate. The combination of biochemical and metabolomic analysis revealed NAT is a novel metabolite of ethanol and a potential biomarker of hyperacetatemia
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