Pyrrole–Hemoglobin Adducts, a More Feasible Potential Biomarker of Pyrrolizidine Alkaloid Exposure

Abstract

Pyrrolizidine alkaloids (PAs) are naturally occurring phytotoxins widely distributed in about 3% of flowering plants. The formation of PA-derived pyrrole–protein adducts is considered as a primary trigger initiating PA-induced hepatotoxicity. The present study aims to (i) further validate our previous established derivatization method using acidified ethanolic AgNO3 for the analysis of pyrrole–protein adducts and (ii) apply this method to characterize the binding tendency, dose–response, and elimination kinetics of pyrrole–protein adducts in blood samples. Two pyrrole–amino acid conjugates, (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)–cysteine (7-cysteine-DHP) and 9-histidine-DHP, were synthesized and used to demonstrate that acidified ethanolic AgNO3 derivatization can cleave both S-linkage and N-linkage of pyrrole–protein adducts. Subsequently, using precolumn AgNO3 derivatization followed by ultra-high-pressure liquid chromatography/mass spectrometry analysis, we quantified pyrrole–protein adducts in monocrotaline-treated rat blood protein fractions, including hemoglobin (Hb), plasma, albumin, and plasma residual protein fractions, and found that the amount of pyrrole–Hb adducts was significantly higher than that in all plasma fractions. Moreover, elimination half-life of pyrrole–Hb adducts was also significantly longer than pyrrole–protein adducts in plasma fractions (12.08 vs 2.54–2.93 days). In addition, we also tested blood samples obtained from five PA-induced liver injury patients and found that the amount of pyrrole–protein adducts in blood cells was also remarkably higher than that in plasma. In conclusion, our findings for the first time confirmed that the AgNO3 derivatization method could be used to measure both S- and N-linked pyrrole–protein adducts and also suggested that pyrrole–Hb adducts with remarkably higher level and longer life span could be a better biomarker of PA exposure.

Developing urinary pyrrole–amino acid adducts as non-invasive biomarkers for identifying pyrrolizidine alkaloids-induced liver injury in human

“…Since then, over 15,000 acute PA-poisoning cases with obvious clinical symptoms have been documented worldwide in many regions/countries, including Afghanistan, China, Germany, Hong Kong, India, Jamaica, South Africa, Switzerland, the United States, etc. (Lin et al 2011 ; Ma et al 2019 ; Ruan et al 2015 ; Zhu et al 2020 ). Nevertheless, the diagnosis of the majority of these reported poisoning cases was based on typical symptoms of HSOS with either known history of exposure to PA-producing herbs or PA-contaminated foodstuffs or through retrospective investigations to confirm the PA exposure.…”

Metabolism-mediated cytotoxicity and genotoxicity of pyrrolizidine alkaloids

“…Further study identified pyrrole-lysine adducts in PA-treated mice, which supported the formation of PPAs in vivo (Li et al 2016). Other proteinogenic amino acids, such as cysteine, valine, and histidine were further found to be capable of binding to the pyrrole moiety through chemical synthesis in the in vitro or in vivo models (Estep et al 1990;He et al 2016aHe et al , b, 2017Ma et al 2019;Xia et al 2015;Zhao et al 2014). All these results support that both DHPAs and DHP possessing bi-functional electrophilic substituents at C7 and C9 positions of the core pyrrole moiety are capable of alkylating to the nucleophilic sites at the -SH and -NH functional groups of the target proteins, leading to the formation of PPAs (Fig.…”

“…DHPAs can rapidly eject a carboxylate leaving group from C1-acyloxymethyl or C7-acyloxy substituents and form intermediates with carbocation. Such iminium intermediates are highly reactive with half-lives of a few seconds in water (Mattocks and Jukes 1990) and capable of alkylating cellular constituents such as proteins and DNA (Ma et al 2019;Xia et al 2013). DHPAs can be further hydrolyzed to ( ±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), which is more water-soluble and less reactive compared with DHPAs but still displays a moderate level of alkylating activity; hence both DHPAs and DHP are considered as reactive metabolites of PAs (Mattocks 1986;Robertson 1982).…”

“…Recently, Lin's group revealed that among different blood fractions tested, the hemoglobin fraction contained the highest level of PPAs, specifically pyrrole-hemoglobin adducts (PHAs), and possessed remarkably prolonged persistence in the blood (Ma et al 2019). Through successful application in 43 PA-ILI patients, PHAs were demonstrated as the more sensitive and reliable PA exposure biomarker comparing with other PPAs such as pyrrole-plasma protein adducts Fig.…”

“…PAs are biologically inactive and require metabolic activation to exert toxicity. Catalyzed by cytochrome P450 enzymes (CYPs) in the liver, PAs are metabolized into their reactive metabolites, which can (1) bind to cellular proteins to form pyrrole-protein adducts (PPAs) and cause cytotoxicity (Ma et al 2019(Ma et al , 2020Ruan et al 2015);…”