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.
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.
Acrylamide forms in many commonly consumed foods. In animals, acrylamide causes tumors, neurotoxicity, developmental and reproductive effects. Acrylamide crosses the placenta and has been associated ...with restriction of intrauterine growth and certain cancers. The impact on human health is poorly understood and it is impossible to say what level of dietary exposure to acrylamide can be deemed safe as the assessment of exposure is uncertain. The determination of hemoglobin (Hb) adducts from acrylamide is increasingly being used to improve the exposure assessment of acrylamide. We aim to outline the literature on Hb adduct levels from acrylamide in humans and discuss methodological issues and research gaps. A total of 86 studies of 27,966 individuals from 19 countries were reviewed. Adduct levels were highest in occupationally exposed individuals and smokers. Levels ranged widely from 3 to 210 pmol/g Hb in non-smokers from the general population and this wide range suggests that dietary exposure to acrylamide varies largely. Non-smokers from the US and Canada had slightly higher levels as compared with non-smokers from elsewhere, but differences within studies were larger than between studies. Large studies with exposure assessment of acrylamide and related adduct forming compounds from diet during early-life are encouraged for the evaluation of health effects.
•We reviewed 86 studies on Hb adducts from acrylamide measured in 27,966 humans from 19 countries.•Adduct levels were highest in occupationally exposed individuals and smokers.•The adduct levels were on average three-fold higher in smokers as compared to non-smokers.•The wide adduct range in non-smokers suggests that dietary acrylamide exposure can be reduced.•Studies on health effects of early-life exposure to acrylamide and related exposures are needed.
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, βSer72, βThr84, βThr87, βSer89, βHis92, βCys93, βCys112, βThr123, and βHis143 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, βCys93, βHis92, and βSer72 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, βSer44, βThr84, and βHis92 were verified in targeted mass spectrometry experiments. Similarly, exposure of human blood to ultraviolet radiation produced 4-hydroxybenzyl adducts at αHis20, αTyr24, αTyr42, αHis45, βSer44, βThr84, and βSer89. 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.
Electrophilically reactive compounds have the ability to form adducts with nucleophilic sites in DNA and proteins, constituting a risk for toxic effects. Mass spectrometric detection of adducts to ...N-terminal valine in hemoglobin (Hb) after detachment by modified Edman degradation procedures is one approach for in vivo monitoring of exposure to electrophilic compounds/metabolites. So far, applications have been limited to one or a few selected reactive species, such as acrylamide and its metabolite glycidamide. This article presents a novel screening strategy for unknown Hb adducts to be used as a basis for an adductomic approach. The method is based on a modified Edman procedure, FIRE, specifically developed for LC–MS/MS analysis of N-terminal valine adducts in Hb detached as fluorescein thiohydantoin (FTH) derivatives. The aim is to detect and identify a priori unknown Hb adducts in human blood samples. Screening of valine adducts was performed by stepwise scanning of precursor ions in small mass increments, monitoring four fragments common for the FTH derivative of valine with different N-substitutions in the multiple-reaction mode, covering a mass range of 135 Da (m/z 503–638). Samples from six smokers and six nonsmokers were analyzed. Control experiments were performed to compare these results with known adducts and to check for artifactual formation of adducts. In all samples of smokers and nonsmokers, seven adducts were identified, of which six have previously been studied. Nineteen unknown adducts were observed, and 14 of those exhibited fragmentation patterns similar to earlier studied FTH derivatives of adducts to valine. Identification of the unknown adducts will be the focus of future work. The presented methodology is a promising screening tool using Hb adducts to indicate exposure to potentially toxic electrophilic compounds and metabolites.
Electrophilic compounds have the ability to form adducts with nucleophilic sites in proteins and DNA in tissues, and thereby constitute risks for toxic effects. Adductomic approaches are developed ...for systematic screening of adducts to DNA and blood proteins, with the aim to detect unknown internal exposures to electrophiles. In a previous adductomic screening of adducts to N-terminals in hemoglobin, using LC-MS/MS, 19 unknown adducts were detected in addition to seven previously identified adducts. The present paper describes the identification of four of these unknown adducts, as well as the strategy used to identify them. Using LC-MS data from the screening, hypotheses about adduct identities were formulated: probable precursor electrophiles with matching molecular weights were suggested based on the molecular weights of the modifications and the retention times of the analytes, in combination with comparisons of theoretical Log P calculations and databases. Reference adducts were generated by incubation of blood samples with the hypothesized precursor electrophiles. The four identified precursor electrophiles, corresponding to the observed unknown adducts, were glyoxal, methylglyoxal, acrylic acid and 1-octen-3-one. Possible origins/exposure sources and toxicological information concerning the electrophilic precursors are discussed. The identified adducts could be explored as possible biomarkers for exposure.
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•Adductome LC-MS/MS data was used for systematic identification of four unknown hemoglobin adducts.•Identifications were based on adductome data combined with databases and Log P calculations.•Glyoxal, methylglyoxal, acrylic acid, and 1-octen-3-one were identified as precursor electrophiles.•The strategy for adduct identification is potentially useful for other adductomic studies.
An Adductomic Approach to Identify Electrophiles In Vivo Carlsson, Henrik; Törnqvist, Margareta
Basic & clinical pharmacology & toxicology,
September 2017, 2017-Sep, 2017-09-00, 20170901, Letnik:
121, Številka:
S3
Journal Article
Recenzirano
Odprti dostop
Human beings are exposed to many reactive electrophiles, both formed endogenously and from exogenous exposures. Such compounds could react with cellular biomolecules and form stable reaction ...products, adducts, at nucleophilic sites in proteins and DNA, constituting a risk for toxic effects. Adductomic approaches aim to study the totality of adducts, to specific biomolecules, by mass spectrometric screening. This Mini‐Review focuses on the development and application of an adductomic approach for the screening of unknown adducts to N‐terminal valine (Val) in haemoglobin (Hb) by liquid chromatography tandem mass spectrometry (LC‐MS/MS). The approach is based on the FIRE procedure, a modified Edman procedure for the analysis of adducts to N‐terminal Val in Hb by LC‐MS/MS. In the first application of the approach, samples from 12 smokers/non‐smokers were screened for Hb adducts, and six previously identified adducts and 20 unknown adducts were detected. To confirm the observation of the detected unknown adducts, targeted screenings were performed in larger sets of blood samples (n = 50–120) from human cohorts. The majority of the previously detected unknown adducts was found in all analysed samples, with large interindividual variations in adduct levels. For structural identification of unknown adducts, a strategy using adductome LC‐MS/MS data was formulated and applied. Six identified adducts correspond to ethylation and the precursor electrophiles ethyl vinyl ketone, glyoxal, methylglyoxal, acrylic acid and 1‐octen‐3‐one. The observation of these adducts in human blood motivate further studies to evaluate possible contributions to health risks, as well as their potential as biomarkers of exposure.
Here we evaluate a multiplicative (relative) risk model for improved cancer risk estimation of genotoxic compounds. According to this model, cancer risk is proportional to the background tumor ...incidence and to the internal dose of the genotoxic compound. Furthermore, the relative risk coefficient per internal dose is considered to be approximately the same across tumor sites, sex, and species.
In the present study, we demonstrate that the relative risk model is valid for cancer risk estimation of glycidol, a common food contaminant. Published tumor data from glycidol carcinogenicity studies in mice and rats were evaluated in combination with internal dose estimates from hemoglobin adduct measurements in blood from mice and rats treated with glycidol in short-term studies. A good agreement between predicted and observed tumor incidence in responding sites was demonstrated in the animals, supporting a relative risk coefficient that is independent of tumor site, sex, and species. There was no significant difference between the risk coefficients for mice (5.1% per mMh) and rats (5.4% per mMh) when considering internal doses of glycidol. Altogether, this mechanism-based risk model gives a reliable risk coefficient, which then was extrapolated to humans considering internal dose, and background cancer incidence.
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•Glycidol animal cancer test data were evaluated with a multiplicative risk model.•In vivo dosimetry of glycidol improved the estimation of the cancer risk coefficient.•A relative cancer risk coefficient for glycidol common for rat and mouse was derived.•The relative risk coefficient was approximately the same for responding tumor sites.•The relative risk coefficient was extrapolated to human exposure levels.
Acrylamide is a probable human carcinogen with widespread exposure via food. The present study compared acrylamide intake measurements obtained from haemoglobin adduct levels and self-registered ...dietary consumption data in a group of 144 Norwegian healthy adults. Acrylamide adducts to N-terminal valine in haemoglobin were measured and used to estimate the intake via the internal dose approach which showed a median (interquartile range) of 0.24 (0.19–0.30) μg/kg bw/day. Data from weighed food records and food frequency questionnaires from the same individuals were used for probabilistic modelling of the intake of acrylamide. The median acrylamide intake was calculated to be 0.26 (0.16–0.39) and 0.30 (0.23–0.39) μg/kg bw/day, respectively from the two sources of self-registered dietary consumption data. Overall, a relatively good agreement was observed between the methods in pairwise comparison in Bland-Altman plots, with the methods disagreeing with 7% or less of the values. The intake estimates obtained with the two dietary consumption methods and one biomarker method are in line with earlier dietary estimates in the Norwegian population. The Margin of Exposure indicate a possible health risk concern from dietary acrylamide. This is the first study with a comparison in the same individuals of acrylamide intake estimates obtained with these methods.
In this study 3-monochloropropane-1,2-diol (3-MCPD), a compound that appears as contaminant in refined cooking oils, has been studied with regard to genotoxicity in vivo (mice) with simultaneous ...measurement of internal dose using state-of-the-art methodologies. Genotoxicity (chromosomal aberrations) was measured by flow cytometry with dual lasers as the frequency of micronuclei in erythrocytes in peripheral blood from BalbC mice intraperitoneally exposed to 3-MCPD (0, 50, 75, 100, 125 mg/kg). The internal doses of 3-MCPD in the mice were calculated from N-(2,3-dihydroxypropyl)-valine adducts to hemoglobin (Hb), quantified at very low levels by high-resolution mass spectrometry.
Convincing evidence for absence of genotoxic potency in correlation to measured internal doses in the mice was demonstrated, despite relatively high administered doses of 3-MCPD. The results are discussed in relation to another food contaminant that is formed as ester in parallel to 3-MCPD esters in oil processing, i.e. glycidol, which has been studied previously by us in a similar experimental setup. Glycidol has been shown to be genotoxic, and in addition to have ca. 1000 times higher rate of adduct formation compared to that observed for 3-MCPD. The conclusion is that at simultaneous exposure to 3-MCPD and glycidol the concern about genotoxicity would be glycidol.
•Convincing evidence for no genotoxicity by 3-MCPD in mice, as monitored by the frequency of micronuclei in erythrocytes.•Hemoglobin (Hb) adducts of 3-MCPD were quantified in the treated mice and showed a linear increase.•A true dose-response was obtained from the frequency of micronuclei related to internal doses, calculated from Hb adducts.
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