Mass Spectral Analyses of Hydroxymethylvinyl Ketone-Hemoglobin Adducts Formed after in Vivo Exposure of Sprague−Dawley Rats to 3-Butene-1,2-diol

Abstract

3-Butene-1,2-diol (BDD), a known in vivo metabolite of 1,3-butadiene, is oxidized to a reactive Michael acceptor, hydroxymethylvinyl ketone (HMVK). Previously, we characterized formation of three HMVK-amino acid monoadducts when HMVK was incubated in vitro with N-acetyl-L-cysteine (NAC), L-valinamide, and N-acetyl-L-lysine (NAL) at physiological conditions. One HMVK-NAL cyclic diadduct (cyclic diadduct 1) also formed by sequential Michael addition reactions of two HMVK molecules with the ε-amino group of NAL followed by enolization and cyclization. Loss of a water molecule and autooxidation converts cyclic diadduct 1 to a more stable cyclic diadduct 2. In the present study, we used multiple mass spectrometry techniques to investigate formation of HMVK adducts with nucleophilic residues of Hb in vivo after dosing Sprague-Dawley (SD) rats with 25 and 200 mg/kg BDD. Trypsin digested globin peptides with mass shifts consistent with the presence of HMVK monoadducts and cyclic diadducts were detected by LC/ESI-QTOF/MS with all rats given BDD. Use of MALDI/FTICR provided further evidence for formation of HMVK monoadducts and cyclic diadducts and use of LC/MS/MS provided unequivocal evidence for adduction of HMVK with Cys125 of globin β chains. Because BDD can also be oxidized to 1,2-dihydroxy-3,4-epoxybutane (EBD), formation of N2-(2,3,4-trihydroxybutyl) (THB)-Hb adducts was also investigated in rats given BDD and several peptides modified by THB were detected. However, because HMVK incubations with red blood cells in vitro also led to detection of THB-Hb adducts, the THB adducts formed in vivo could be attributed to formation of HMVK, EBD, or both. Collectively, the results provide new insights into the reaction of HMVK with proteins.

Globin Monoadducts and Cross-Links Provide Evidence for the Presence of S-(1,2-Dichlorovinyl)-l-cysteine Sulfoxide, Chlorothioketene, and 2-Chlorothionoacetyl Chloride in the Circulation in Rats Administered S-(1,2-Dichlorovinyl)-l-cysteine

“…Hb adducts are used for biomonitoring of toxicant exposure in human epidemiological studies − and to elucidate the relative contributions of different metabolic pathways in cases where a toxicant can yield multiple reactive metabolites − . The long life span of erythrocytes (corresponds to turnover of Hb; 120 days for humans and 63 days for rats) and the stability of adducts allow detection of short-lived reactive metabolites present in the circulation at low levels of toxicant exposure over an extended period of time.…”

Identification and Quantification of 1-Hydroxybutene-2-yl Mercapturic Acid in Human Urine by UPLC- HILIC-MS/MS as a Novel Biomarker for 1,3-Butadiene Exposure

“…Next, this monoepoxide is conjugated to glutathione (GSH, γ- l -glutamyl- l -cysteinylglycine) by glutathione S -transferases (GSTs) to yield the two isomeric intermediate compounds (1-hydroxy-3-butene-2-yl)-GSH and (2-hydroxy-3-butene-1-yl)-GSH. Further enzymatic reactions of γ-glutamyltranspeptidase (GGT), a cysteinylglycine dipeptidase (DPEP), and cysteine S-conjugate N -acetyltransferase (NAT8) stepwisely degrade these conjugates finally leading to two isomers of monohydroxybutenyl mercapturic acid, (1-hydroxy-3-butene-2-yl)- N -acetyl- l -cysteine (1-MHBMA) and (2-hydroxy-3-butene-1-yl)- N -acetyl- l -cysteine (2-MHBMA), which are excreted in urine. , Besides these metabolites, BD could also be converted to dihydroxybutyl mercapturic acid (DHBMA) and trihydroxybutyl mercapturic acid (THBMA) as well as form various hemoglobin and DNA adducts. − The genotoxic effects of BD are attributed to its DNA-reactive epoxide metabolites …”

Alcohol Dehydrogenase- and Rat Liver Cytosol-Dependent Bioactivation of 1-Chloro-2-hydroxy-3-butene to 1-Chloro-3-buten-2-one, a Bifunctional Alkylating Agent

“…33 As a reactive Michael acceptor, HMVK exhibited high reactivity towards nucleophilic amino acids, 42 hemoglobin, 43 and nucleosides and DNA. 44 Thus, it is expected that CHB can also be biotransformed to CBO in vivo , which can react with a variety of biomacromolecules.…”

“…Evidence for BDD oxidation to HMVK in mice and rats in vivo 41 was obtained by the detection of the urinary metabolite 4-(N-acetyl-L-cystein-S-yl)-1,2-dihydroxybutane (the mercapturic acid of HMVK−GSH conjugate) 32 and detection of HMVK−globin adducts in rats and mice given BDD. 33 As a reactive Michael acceptor, HMVK exhibited high reactivity toward nucleophilic amino acids, 42 hemoglobin, 43 and nucleosides and DNA. 44 Thus, it is expected that CHB can also be biotransformed to CBO in vivo, which can react with a variety of biomacromolecules.…”

“…Support for this hypothesis is provided by our previous demonstration that 3-butene-1,2-diol (BDD) can be metabolized by ADHs and cytochromes P450 both in vitro and in vivo to yield the Michael acceptor hydroxymethylvinyl ketone (HMVK). 29–33 We postulated that CHB could similarly be transformed to 1-chloro-3-buten-2-one (CBO, 2 ) by ADHs (Scheme 1). Based on its chemical structure, CBO is expected to act as a bifunctional alkylating agent.…”