ARTICLE pubs.acs.org/crt
Analysis of Hemoglobin Adducts from Acrylamide, Glycidamide, and Ethylene Oxide in Paired Mother/Cord Blood Samples from Denmark Hans von Stedingk,† Anna C. Vikstr€om,‡ Per Rydberg,† Marie Pedersen,‡,|| Jeanette K. S. Nielsen,‡ Dan Segerb€ack,§ Lisbeth E. Knudsen,*,‡ and Margareta T€ornqvist*,† †
Department of Materials and Environmental Chemistry, Environmental Chemistry Unit, Arrhenius Laboratory, Stockholm University, SE-106 91 Stockholm, Sweden ‡ Section of Environmental Health, Department of Public Health, University of Copenhagen, Oester Farimagsgade 5A, 1353 K Copenhagen, Denmark § Department of Biosciences and Nutrition, Karolinska Institute, Novum, SE-141 83 Huddinge, Sweden ABSTRACT: The knowledge about fetal exposure to acrylamide/glycidamide from the maternal exposure through food is limited. Acrylamide, glycidamide, and ethylene oxide are electrophiles and form adducts with hemoglobin (Hb), which could be used for in vivo dose measurement. In this study, a method for analysis of Hb adducts by liquid chromatography mass spectrometry, the adduct FIRE procedure, was applied to measurements of adducts from these compounds in maternal blood samples (n = 87) and umbilical cord blood samples (n = 219). The adduct levels from the three compounds, acrylamide, glycidamide, and ethylene oxide, were increased in tobacco smokers. Highly significant correlations were found between cord and maternal blood with regard to measured adduct levels of the three compounds. The mean cord/ maternal hemoglobin adduct level ratios were 0.48 (range 0.27 0.86) for acrylamide, 0.38 (range 0.20 0.73) for glycidamide, and 0.43 (range 0.17 1.34) for ethylene oxide. In vitro studies with acrylamide and glycidamide showed a lower (0.38 0.48) rate of adduct formation with Hb in cord blood than with Hb in maternal blood, which is compatible with the structural differences in fetal and adult Hb. Together, these results indicate a similar life span of fetal and maternal erythrocytes. The results showed that the in vivo dose in fetal and maternal blood is about the same and that the placenta gives negligible protection of the fetus to exposure from the investigated compounds. A trend of higher levels of the measured adducts in cord blood with gestational age was observed, which may reflect the gestational age-related change of the cord blood Hb composition toward a higher content of adult Hb. The results suggest that the Hb adduct levels measured in cord blood reflect the exposure to the fetus during the third trimester. The evaluation of the new analytical method showed that it is suitable for monitoring of background exposures of the investigated electrophilic compounds in large population studies.
’ INTRODUCTION Exposure to toxic chemicals in utero is a particular risk due to the vulnerability and the critical periods during fetal development. There is increasing knowledge that in utero exposure to certain chemicals gives rise to adverse health effects.1,2 With regard to carcinogens, recent evaluations have led to a recommendation that a default potency factor of 10 should be used to adjust for the increased susceptibility for exposure during the prenatal period (third trimester) or young age compared to exposure during adult age (reviewed in ref 3). During the last decades, evidence has accumulated that the human placenta does not give complete protection for exposure to toxic compounds.4,5 In the present study, hemoglobin (Hb) adducts are used to clarify the exposure to the fetus via placental transfer of a few electrophilically reactive and genotoxic compounds. Ehrenberg and co-workers suggested in 1974 that in vivo doses, i.e., concentration over time of electrophilic compounds/ metabolites, could be measured through protein adducts for the purpose of risk assessment of genotoxic compounds.6 Hb is r 2011 American Chemical Society
advantageous as a monitor molecule as adducts are accumulated over the life-span of the erythrocytes, and Hb adducts could accordingly be used to measure exposure up to 4 months back in time in adult humans. During chronic exposure, the adduct level reaches a steady-state, which thus reflects the average in vivo dose.7 A facile and sensitive method for analysis of Hb adducts by a modified Edman degradation procedure was described in 1986.8 This so-called N-alkyl Edman method has been used to measure simple alkylating agents/metabolites in studies of occupational exposure and of exposure from tobacco smoking in humans, and the method has further been used in studies to identify background exposure in the general population.7,9 This method uses a fluorinated Edman reagent (pentafluorophenyl isothiocyanate) to specifically detach adducts with the N-terminal amino acid in Hb (valine in the α- and β-chains in adult human Hb), which gives a derivative of N-substituted valines Received: July 13, 2011 Published: September 01, 2011 1957
dx.doi.org/10.1021/tx200284u | Chem. Res. Toxicol. 2011, 24, 1957–1965
Chemical Research in Toxicology suitable for analysis by gas chromatography mass spectrometry (GC/MS). With the aim to achieve a faster and less labor intensive method for a broader application range, the adduct FIRE procedure (fluorescein isothiocyanate R Edman) was developed, which uses an Edman reagent more suitable for liquid chromatography/mass spectrometry analysis (LC/MS).10 12 In the present study, this method is applied to measure exposure to acrylamide, glycidamide, and ethylene oxide in mother child pairs. Adduct analysis by the N-alkyl Edman method was the method that was used for the detection and identification of the generally occurring exposure to acrylamide in nonsmokers.13,14 This background exposure was shown to originate from acrylamide formed during the processing of carbohydrate-rich food at high temperatures.14 16 Cigarette smoke is another source of acrylamide,17 giving rise to elevated Hb adduct levels.13,18 Acrylamide is classified as a probable human carcinogen19 and has been shown to be both a neuro- and reproductive toxicant.20,21 Acrylamide is biotransformed to the genotoxic metabolite glycidamide.22 24 The Hb adduct corresponding to ethylene oxide, N-(2-hydroxyethyl)valine, has also been shown to generally occur in the population. 25,26 Ethylene oxide is formed as a metabolite of ethylene.27,28 The compound is classified as carcinogenic to humans29 and has shown reproductive toxicity both in animals30 and humans.31 Ethylene/ethylene oxide is formed in normal metabolism, and studies in mice have shown an influence of dietary fat and intestinal flora on endogenous production (reviewed in ref 26). Compared to endogenous formation, the exposure to ethylene in urban air pollution is estimated to give a minor contribution to the level of N-(2-hydroxyethyl)valine.27 Cigarette smokers have clearly increased levels of N-(2-hydroxyethyl)valine, and the adduct has been suggested as a marker for exposure to cigarette smoke from both active and passive smoking.18,32,33 Ex vivo experiments with perfusions of the human placenta have shown that both acrylamide and glycidamide pass the placental tissue.34,35 A few studies were performed with the measurement of Hb adducts from acrylamide36 and ethylene oxide37,38 in blood from pregnant women and cord blood or blood from newborns. Maternal adduct levels were twice as high as those observed in cord blood/blood from newborns. Possible explanations could be an effect of different N-terminal amino acids in fetal versus adult Hb chains37,38 and/or shorter lifetime of the fetal erythrocytes.36 The aim of this study was to characterize the Hb adduct levels from acrylamide and its metabolite glycidamide from dietary intake, and from ethylene oxide primarily as a control of exposure to tobacco smoke, in mother newborns from Denmark. Methodological aspects concerning Hb adduct measurements for assessment and comparison of in vivo dose in the mother and her fetus from exposure to the electrophilic agents were investigated. Furthermore, the applicability of the new adduct FIRE procedure to a population study was evaluated.
’ MATERIAL AND METHODS Caution: The following chemicals are hazardous and should be handled carefully: fluoresceine-5-isothiocyanate, acrylamide, and glycidamide. Chemicals. Fluoresceine-5-isothiocyanate (FITC isomer I,
