Role of covalent modification by hepatic aldehydes in dictamnine-induced liver injury.

Dictamnine is a representative furan-containing hepatotoxic compound. Administration of dictamnine caused acute liver injury in mice and the metabolic activation of furan to reactive epoxy intermediate was responsible for the hepatotoxicity. This study aimed to characterize the protein adduction by endogenous hepatic aldehydes and investigate its role in dictamnine-induced hepatotoxicity. In the liver sample of dictamnine-treated mice, the protein adduction by five aldehydes was characterized as lysine residue-aldehyde adducts using high-resolution UPLC-Q/Orbitrap MS after exhaustive proteolytic digestion. The levels of protein adduct were increased at 2-3 h after the treatment with dictamnine. The formation of protein adduction increased with increasing doses of dictamnine. Inhibition of the bioactivation by CYP3A inhibitor ketoconazole prevented the protein adduction. Treatment with 2,3-dihydro-dictamnine, an analog of dictamnine that was unable to form the epoxy intermediate, did not lead to an increase in protein adduction. Application of aldehyde dehydrogenase-2 activator ALDA-1 or nucleophilic trapping reagent N-acetyl-L-lysine significantly reduced the protein adduction and attenuated dictamnine-induced liver injury without affecting the bioactivation. In conclusion, the metabolic activation of the furan ring of dictamnine resulted in the protein adduction by multiple hepatic aldehydes and the protein modification played a crucial role in dictamnine-induced liver injury.

Widespread occurrence of in-source fragmentation in the analysis of natural compounds by liquid chromatography-electrospray ionization mass spectrometry.

RATIONALE: The in-source fragmentation (ISF) of analyte or co-eluting substances produces unintentional fragment ions, which hampers identification and quantification by liquid chromatography-mass spectrometry (LC/MS). Natural compounds derived from plants also contain fragile moieties that may undergo ISF. However, the characteristics of ISF of natural compounds in LC/MS are still unclear. METHODS: The ISF behavior of 214 natural compounds was assayed in LC with Q/orbitrap MS in electrospray ionization (ESI) mode and the extent of ISF was evaluated. RESULTS: Up to 82% of tested compounds underwent ISF and half of the tested natural compounds that contain more than one fragile moiety underwent successive and severe ISF to generate serial structurally related ISF products. The major ISF-altering moieties for natural compounds were hydroxyl, lactone, glycosyl and ether, resulting in neutral loss of H2 O or CO, deglycosylation or cleavage of ether bond, respectively. Some compounds such as terpenoids underwent severe ISF and less than 1% parent form can be observed. For natural compounds, ISF products with similar structures are more likely to cause interference in analysis because the ISF products may share identical mass-to-charge ratio and similar MS2 fragmentation patterns with precursor ions of the homologs in plants. Furthermore, severe ISF may cause a false negative in the identification of the parent form. CONCLUSIONS: In summary, ISF was a highly frequent phenomenon for analysis of natural compounds by LC/ESI-MS, and extensive and successive ISF of natural products may cause misannotation and misidentification with homologs in plants. The study should raise awareness of ISF interference during the analysis of natural compounds.

Feature MS fragments-based method for identification of toxic furanoids in biological samples.

Numerous furan-containing compounds have been reported to be toxic. The toxicity may be attributed to the metabolic activation of the furan ring to cis-enediones. Identification of unknown furans that undergo bioactivation is challenging. Here, we present a novel approach that enables non-targeted profiling of bioactivation of unknown furanoids both in vitro and in vivo. Cyclic pyrrole-glutathione conjugate was the predominant product of cis-enediones with glutathione. The shared glutathione substructure of conjugates was capable of generating four constant and signature fragments under collision-induced dissociation (CID) in the mass spectrometer, including neutral loss fragments 103.0269 Da and 146.0691 Da and product ions at m/z 130.0499 and 177.0328. The unique structure and high abundance of conjugates in combination with the consistency and specificity of CID fragmentation brought extraordinarily high selectivity and reliability for the four fragments as a fingerprint of bioactivated furanoids. The bioactivated furanoids can be identified by screening the four fragments in high-resolution MS/MS datasets using the neutral loss filtering and diagnostic fragmentation filtering of data post-acquisition software MZmine. The simultaneous formation of four individual signal points in the filtering channel with the same precursor ion and retention time was assigned to be furanoids. The method has been rigorously validated. In the pooled urine samples from nine model furanoids-treated mice, nine cis-enediones from the parent furanoids and two from furanoid metabolites were accurately detected and identified. The method showed great performance in non-targeted profiling bioactivated furanoids and their metabolites in urine samples of herbal extract-treated mice.