Mass Spectrometry-Based Strategies for Assessing Human Exposure Using Hemoglobin Adductomics.

Hemoglobin (Hb) adducts are widely used in human biomonitoring due to the high abundance of hemoglobin in human blood, its reactivity toward electrophiles, and adducted protein stability for up to 120 days. In the present paper, we compared three methods of analysis of hemoglobin adducts: mass spectrometry of derivatized N-terminal Val adducts, mass spectrometry of N-terminal adducted hemoglobin peptides, and limited proteolysis mass spectrometry . Blood from human donors was incubated with a selection of contact allergens and other electrophiles, after which hemoglobin was isolated and subjected to three analysis methods. We found that the FIRE method was able to detect and reliably quantify N-terminal adducts of acrylamide, acrylic acid, glycidic acid, and 2,3-epoxypropyl phenyl ether (PGE), but it was less efficient for 2-methyleneglutaronitrile (2-MGN) and failed to detect 1-chloro-2,4-dinitrobenzene (DNCB). By contrast, bottom-up proteomics was able to determine the presence of adducts from all six electrophiles at both the N-terminus and reactive hemoglobin side chains. Limited proteolysis mass spectrometry, studied for four contact allergens (three electrophiles and a metal salt), was able to determine the presence of covalent hemoglobin adducts with one of the three electrophiles (DNCB) and coordination complexation with the nickel salt. Together, these approaches represent complementary tools in the study of the hemoglobin adductome.

Pathways to Identify Electrophiles In Vivo Using Hemoglobin Adducts: Hydroxypropanoic Acid Valine Adduct and Its Possible Precursors.

Analytical methods and tools for the characterization of the human exposome by untargeted mass spectrometry approaches are advancing rapidly. Adductomics methods have been developed for untargeted screening of short-lived electrophiles, in the form of adducts to proteins or DNA, in vivo. The identification of an adduct and its precursor electrophile in the blood is more complex than that of stable chemicals. The present work aims to illustrate procedures for the identification of an adduct to N-terminal valine in hemoglobin detected with adductomics, and pathways for the tracing of its precursor and possible exposure sources. Identification of the adduct proceeded via preparation and characterization of standards of adduct analytes. Possible precursor(s) and exposure sources were investigated by measurements in blood of adduct formation by precursors in vitro and adduct levels in vivo. The adduct was identified as hydroxypropanoic acid valine (HPA-Val) by verification with a synthesized reference. The HPA-Val was measured together with other adducts (from acrylamide, glycidamide, glycidol, and acrylic acid) in human blood (n = 51, schoolchildren). The HPA-Val levels ranged between 6 and 76 pmol/g hemoglobin. The analysis of reference samples from humans and rodents showed that the HPA-Val adduct was observed in all studied samples. No correlation of the HPA-Val level with the other studied adducts was observed in humans, nor was an increase in tobacco smokers observed. A small increase was observed in rodents exposed to glycidol. The formation of the HPA-Val adduct upon incubation of blood with glycidic acid (an epoxide) was shown. The relatively high adduct levels observed in vivo in relation to the measured reactivity of the epoxide, and the fact that the epoxide is not described as naturally occurring, suggest that glycidic acid is not the only precursor of the HPA-Val adduct identified in vivo. Another endogenous electrophile is suspected to contribute to the in vivo HPA-Val adduct level.

Novel 4-Hydroxybenzyl Adducts in Human Hemoglobin: Structures and Mechanisms of Formation.

Humans are exposed to large numbers of electrophiles from their diet, the environment, and endogenous physiological processes. Adducts formed at the N-terminal valine of hemoglobin are often used as biomarkers of human exposure to electrophilic compounds. We previously reported the formation of hemoglobin N-terminal valine adducts (added mass, 106.042 Da) in the blood of human smokers and nonsmokers and identified their structure as 4-hydroxybenzyl-Val. In the present work, mass spectrometry-based proteomics was utilized to identify additional sites for 4-hydroxybenzyl adduct formation at internal nucleophilic amino acid side chains within hemoglobin. Hemoglobin isolated from human blood was treated with para-quinone methide (para-QM) followed by global nanoLC-MS/MS and targeted nanoLC-MS/MS to identify amino acid residues containing the 4-hydroxybenzyl modification. Our experiments revealed the formation of 4-hydroxybenzyl adducts at the αHis20, αTyr24, αTyr42, αHis45, ßSer72, ßThr84, ßThr87, ßSer89, ßHis92, ßCys93, ßCys112, ßThr123, and ßHis143 residues (in addition to N-terminal valine) through characteristic MS/MS spectra. These amino acid side chains had variable reactivity toward para-QM with αHis45, αTyr42, ßCys93, ßHis92, and ßSer72 forming the largest numbers of adducts upon exposure to para-QM. Two additional mechanisms for formation of 4-hydroxybenzyl adducts in humans were investigated: exposure to 4-hydroxybenzaldehyde (4-HBA) followed by reduction and UV-mediated reactions of hemoglobin with tyrosine. Exposure of hemoglobin to a 5-fold molar excess of 4-HBA followed by reduction with sodium cyanoborohydride produced 4-hydroxybenzyl adducts at several amino acid side chains of which αHis20, αTyr24, αTyr42, αHis45, ßSer44, ßThr84, and ßHis92 were verified in targeted mass spectrometry experiments. Similarly, exposure of human blood to ultraviolet radiation produced 4-hydroxybenzyl adducts at αHis20, αTyr24, αTyr42, αHis45, ßSer44, ßThr84, and ßSer89. Overall, our results reveal that 4-hydroxybenzyl adducts form at multiple nucleophilic sites of hemoglobin and that para-QM is the most likely source of these adducts in humans.

Adduct levels from benzo[a]pyrenediol epoxide: Relative formation to histidine in serum albumin and to deoxyguanosine in DNA in vitro and in vivo in mice measured by LC/MS-MS methods.

Stable and specific biomacromolecular adducts can be used to measure in vivo doses of reactive compounds. An LC/MS-MS method to measure adducts from the benzo[a]pyrene (BP) metabolite (±)-anti-BP-7,8-diol-9,10-epoxide ((±)-anti-BPDE) to His(146) in serum albumin (SA), earlier evaluated on in vitro alkylated human SA, was tested for its applicability to mouse. It was shown that (+)-anti-BPDE form BPDE-His adducts to mouse SA. The method was applied to samples from BP-exposed mice (100mg/kg of body weight for 1, 3, 7 and 28 days). BPDE-His in SA was close to the limit of quantification and showed the highest level (13fmol/mg) 3 days after exposure. The level was 400 times lower (calculated per gram macromolecule) than earlier measured level of BPDE-adduct to deoxyguanosine (dG) in DNA in the livers. The relative rate of formation of adducts from BPDE with His in SA and with dG in DNA was investigated. Quantification by LC/MS-MS of the adducts in human blood alkylated in vitro with (±)-anti-BPDE showed a 1850 times higher level of BPDE-dG compared to BPDE-His. The specific and stable BPDE-adducts to His in SA are potential biomarkers of in vivo dose of BPDE, though this requires a considerable improved analytical sensitivity of the LC/MS-MS method.

In vivo doses of acrylamide and glycidamide in humans after intake of acrylamide-rich food.

For assessment of cancer risk from acrylamide (AA) exposure through food, the relation between intake from food in humans and the in vivo doses (area under the concentration-time curve, AUC) of AA (AUC-AA) and of its genotoxic metabolite glycidamide (GA) (AUC-GA) is used as a basis for extrapolation between exposure levels and between species. In this study, AA-rich foods were given to nonsmokers: a high intake of 11 µg AA/kg body weight (bw) and day for 4 days or an extra (medium) intake of 2.5 µg AA/kg bw and day for a month. Hemoglobin (Hb)-adduct levels from AA and GA, measured in blood samples donated before and after exposures, were used for calculation of AUC-AA and AUC-GA using reaction rate constants for the adduct formation measured in vitro. Both AA- and GA-adduct levels increased about twofold after the periods with enhanced intake. AUC for the high and medium groups, respectively, in nanomolar hours per microgram AA per kilogram bw, was for AA 212 and 120 and for GA 49 and 21. The AA intake in the high group was better controlled and used for comparisons with other data. The AUCs per exposure dose obtained in the present human study (high group) are in agreement with those previously obtained at 10(2) times higher exposure levels in humans. Furthermore, the values of AUC-AA and AUC-GA are five and two times higher, respectively, than the corresponding values for F344 rats exposed to AA at levels as in published cancer bioassays.