Hans von Stedingk

Birth Weight, Head Circumference, and Prenatal Exposure to Acrylamide from Maternal Diet: The European Prospective Mother–Child Study (NewGeneris)

Background: Acrylamide is a common dietary exposure that crosses the human placenta. It is classified as a probable human carcinogen, and developmental toxicity has been observed in rodents. Objectives: We examined the associations between prenatal exposure to acrylamide and birth outcomes in a prospective European mother–child study. Methods: Hemoglobin (Hb) adducts of acrylamide and its metabolite glycidamide were measured in cord blood (reflecting cumulated exposure in the last months of pregnancy) from 1,101 singleton pregnant women recruited in Denmark, England, Greece, Norway, and Spain during 2006–2010. Maternal diet was estimated through food-frequency questionnaires. Results: Both acrylamide and glycidamide Hb adducts were associated with a statistically significant reduction in birth weight and head circumference. The estimated difference in birth weight for infants in the highest versus lowest quartile of acrylamide Hb adduct levels after adjusting for gestational age and country was –132 g (95% CI: –207, –56); the corresponding difference for head circumference was –0.33 cm (95% CI: –0.61, –0.06). Findings were similar in infants of nonsmokers, were consistent across countries, and remained after adjustment for factors associated with reduced birth weight. Maternal consumption of foods rich in acrylamide, such as fried potatoes, was associated with cord blood acrylamide adduct levels and with reduced birth weight. Conclusions: Dietary exposure to acrylamide was associated with reduced birth weight and head circumference. Consumption of specific foods during pregnancy was associated with higher acrylamide exposure in utero. If confirmed, these findings suggest that dietary intake of acrylamide should be reduced among pregnant women.

Analysis of hemoglobin adducts from acrylamide, glycidamide, and ethylene oxide in paired mother/cord blood samples from Denmark.

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.

Global Gene Expression Analysis in Cord Blood Reveals Gender-Specific Differences in Response to Carcinogenic Exposure In Utero

Background: It has been suggested that fetal carcinogenic exposure might lead to predisposition to develop cancer during childhood or in later life possibly through modulation of the fetal transcriptome. Because gender effects in the incidence of childhood cancers have been described, we hypothesized differences at the transcriptomic level in cord blood between male and female newborns as a consequence of fetal carcinogenic exposure. The objective was to investigate whether transcriptomic responses to dietary genotoxic and nongenotoxic carcinogens show gender-specific mechanisms-of-action relevant for chemical carcinogenesis. Methods: Global gene expression was applied in umbilical cord blood samples, the CALUX-assay was used for measuring dioxin(-like), androgen(-like), and estrogen(-like) internal exposure, and acrylamide–hemoglobin adduct levels were determined by mass spectrometry adduct-FIRE-procedureTM. To link gene expression to an established phenotypic biomarker of cancer risk, micronuclei frequencies were investigated. Results: While exposure levels did not differ between sexes at birth, important gender-specific differences were observed in gene expressions associated with these exposures linked with cell cycle, the immune system and more general cellular processes such as posttranslation. Moreover, oppositely correlating leukemia/lymphoma genes between male and female newborns were identified in relation to the different biomarkers of exposure that might be relevant to male-specific predisposition to develop these cancers in childhood. Conclusions/Impact: This study reveals different transcriptomic responses to environmental carcinogens between the sexes. In particular, male-specific TNF-alpha-NF-kB signaling upon dioxin exposure and activation of the Wnt-pathway in boys upon acrylamide exposure might represent possible mechanistic explanations for gender specificity in the incidence of childhood leukemia. Cancer Epidemiol Biomarkers Prev; 21(10); 1756–67. ©2012 AACR.

Dietary Acrylamide Intake during Pregnancy and Fetal Growth—Results from the Norwegian Mother and Child Cohort Study (MoBa)

Background: Acrylamide has shown developmental and reproductive toxicity in animals, as well as neurotoxic effects in humans with occupational exposures. Because it is widespread in food and can pass through the human placenta, concerns have been raised about potential developmental effects of dietary exposures in humans. Objectives: We assessed associations of prenatal exposure to dietary acrylamide with small for gestational age (SGA) and birth weight. Methods: This study included 50,651 women in the Norwegian Mother and Child Cohort Study (MoBa). Acrylamide exposure assessment was based on intake estimates obtained from a food frequency questionnaire (FFQ), which were compared with hemoglobin (Hb) adduct measurements reflecting acrylamide exposure in a subset of samples (n = 79). Data on infant birth weight and gestational age were obtained from the Medical Birth Registry of Norway. Multivariable regression was used to estimate associations between prenatal acrylamide and birth outcomes. Results: Acrylamide intake during pregnancy was negatively associated with fetal growth. When women in the highest quartile of acrylamide intake were compared with women in the lowest quartile, the multivariable-adjusted odds ratio (OR) for SGA was 1.11 (95% CI: 1.02, 1.21) and the coefficient for birth weight was –25.7 g (95% CI: –35.9, –15.4). Results were similar after excluding mothers who smoked during pregnancy. Maternal acrylamide– and glycidamide–Hb adduct levels were correlated with estimated dietary acrylamide intakes (Spearman correlations = 0.24; 95% CI: 0.02, 0.44; and 0.48; 95% CI: 0.29, 0.63, respectively). Conclusions: Lowering dietary acrylamide intake during pregnancy may improve fetal growth.

Bulky DNA Adducts in Cord Blood, Maternal Fruit-and-Vegetable Consumption, and Birth Weight in a European Mother–Child Study (NewGeneris)

Background: Tobacco-smoke, airborne, and dietary exposures to polycyclic aromatic hydrocarbons (PAHs) have been associated with reduced prenatal growth. Evidence from biomarker-based studies of low-exposed populations is limited. Bulky DNA adducts in cord blood reflect the prenatal effective dose to several genotoxic agents including PAHs. Objectives: We estimated the association between bulky DNA adduct levels and birth weight in a multicenter study and examined modification of this association by maternal intake of fruits and vegetables during pregnancy. Methods: Pregnant women from Denmark, England, Greece, Norway, and Spain were recruited in 2006–2010. Adduct levels were measured by the 32P-postlabeling technique in white blood cells from 229 mothers and 612 newborns. Maternal diet was examined through questionnaires. Results: Adduct levels in maternal and cord blood samples were similar and positively correlated (median, 12.1 vs. 11.4 adducts in 108 nucleotides; Spearman rank correlation coefficient = 0.66, p < 0.001). Cord blood adduct levels were negatively associated with birth weight, with an estimated difference in mean birth weight of –129 g (95% CI: –233, –25 g) for infants in the highest versus lowest tertile of adducts. The negative association with birth weight was limited to births in Norway, Denmark, and England, the countries with the lowest adduct levels, and was more pronounced in births to mothers with low intake of fruits and vegetables (–248 g; 95% CI: –405, –92 g) compared with those with high intake (–58 g; 95% CI: –206, 90 g) Conclusions: Maternal exposure to genotoxic agents that induce the formation of bulky DNA adducts may affect intrauterine growth. Maternal fruit and vegetable consumption may be protective. Citation: Pedersen M, Schoket B, Godschalk RW, Wright J, von Stedingk H, Törnqvist M, Sunyer J, Nielsen JK, Merlo DF, Mendez MA, Meltzer HM, Lukács V, Landström A, Kyrtopoulos SA, Kovács K, Knudsen LE, Haugen M, Hardie LJ, Gützkow KB, Fleming S, Fthenou E, Farmer PB, Espinosa A, Chatzi L, Brunborg G, Brady NJ, Botsivali M, Arab K, Anna L, Alexander J, Agramunt S, Kleinjans JC, Segerbäck D, Kogevinas M. 2013. Bulky DNA adducts in cord blood, maternal fruit-and-vegetable consumption, and birth weight in a European mother–child study (NewGeneris). Environ Health Perspect 121:1200–1206; http://dx.doi.org/10.1289/ehp.1206333

Micronuclei in cord blood lymphocytes and associations with biomarkers of exposure to carcinogens and hormonally active factors, gene polymorphisms, and gene expression: The newgeneris cohort

Background: Leukemia incidence has increased in recent decades among European children, suggesting that early-life environmental exposures play an important role in disease development. Objectives: We investigated the hypothesis that childhood susceptibility may increase as a result of in utero exposure to carcinogens and hormonally acting factors. Using cord blood samples from the NewGeneris cohort, we examined associations between a range of biomarkers of carcinogen exposure and hormonally acting factors with micronuclei (MN) frequency as a proxy measure of cancer risk. Associations with gene expression and genotype were also explored. Methods: DNA and protein adducts, gene expression profiles, circulating hormonally acting factors, and GWAS (genome-wide association study) data were investigated in relation to genomic damage measured by MN frequency in lymphocytes from 623 newborns enrolled between 2006 and 2010 across Europe. Results: Malondialdehyde DNA adducts (M1dG) were associated with increased MN frequency in binucleated lymphocytes (MNBN), and exposure to androgenic, estrogenic, and dioxin-like compounds was associated with MN frequency in mononucleated lymphocytes (MNMONO), although no monotonic exposure–outcome relationship was observed. Lower frequencies of MNBN were associated with a 1-unit increase expression of PDCD11, LATS2, TRIM13, CD28, SMC1A, IL7R, and NIPBL genes. Gene expression was significantly higher in association with the highest versus lowest category of bulky and M1dG–DNA adducts for five and six genes, respectively. Gene expression levels were significantly lower for 11 genes in association with the highest versus lowest category of plasma AR CALUX® (chemically activated luciferase expression for androgens) (8 genes), ERα CALUX® (for estrogens) (2 genes), and DR CALUX® (for dioxins). Several SNPs (single-nucleotide polymorphisms) on chromosome 11 near FOLH1 significantly modified associations between androgen activity and MNBN frequency. Polymorphisms in EPHX1/2 and CYP2E1 were associated with MNBN. Conclusion: We measured in utero exposure to selected environmental carcinogens and circulating hormonally acting factors and detected associations with MN frequency in newborns circulating T lymphocytes. The results highlight mechanisms that may contribute to carcinogen-induced leukemia and require further research. Citation: Merlo DF, Agramunt S, Anna L, Besselink H, Botsivali M, Brady NJ, Ceppi M, Chatzi L, Chen B, Decordier I, Farmer PB, Fleming S, Fontana V, Försti A, Fthenou E, Gallo F, Georgiadis P, Gmuender H, Godschalk RW, Granum B, Hardie LJ, Hemminki K, Hochstenbach K, Knudsen LE, Kogevinas M, Kovács K, Kyrtopoulos SA, Løvik M, Nielsen JK, Nygaard UC, Pedersen M, Rydberg P, Schoket B, Segerbäck D, Singh R, Sunyer J, Törnqvist M, van Loveren H, van Schooten FJ, Vande Loock K, von Stedingk H, Wright J, Kleinjans JC, Kirsch-Volders M, van Delft JHM, NewGeneris Consortium. 2014. Micronuclei in cord blood lymphocytes and associations with biomarkers of exposure to carcinogens and hormonally active factors, gene polymorphisms, and gene expression: The NewGeneris Cohort. Environ Health Perspect 122:193–200; http://dx.doi.org/10.1289/ehp.1206324

LC–MS/MS Screening Strategy for Unknown Adducts to N-Terminal Valine in Hemoglobin Applied to Smokers and Nonsmokers

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.

LC/MS/MS Analysis of N-Terminal Protein Adducts with Improved Sensitivity: A Comparison of Selected Edman Isothiocyanate Reagents.

This study provides a basis for a new and straightforward method for LC/MS/MS-based screening of N-terminal protein adducts. This procedure is denoted the “FIRE procedure” as fluorescein isothiocyanate (FITC) gave superior sensitivity by LC/MS/MS when measuring adducts (R) of electrophilic compounds with a modified Edman procedure. The principles of the FIRE-procedure are that adducts to N-terminal amino acids selectively are detached and measured from of proteins after derivatisation by isothiocyanate Edman reagents. In this study, FITC, 4-N,N-dimethylaminoazobenzene 4′-isothiocyanate (DABITC) and 4-dimethylamino-1-naphthyl isothiocyanate (DNITC) were used to synthesize thiohydantoin analytes from valine and N-methylvaline. The sensitivity by LC/MS/MS was enhanced by up to three orders of magnitude as compared to phenyl isothiocyanate and higher as compared to pentafluorophenyl isothiocyanate. The FITC reagent will enable measurements of low background adduct levels. Synthesized analytes were characterised with, for example, 1H NMR, 13C NMR, LC/MS/MS, and UV.

Methyl vinyl ketone--identification and quantification of adducts to N-terminal valine in human hemoglobin.

Adducts to N-terminal valines in Hb have been shown useful as biomarkers of exposure to electrophilic compounds. Adducts from many compounds have earlier been measured with a modified Edman degradation method using a GC-MS/MS method. A recently developed method, the adduct FIRE procedure™, adopted for analysis by LC-MS/MS, has been applied in this study. With this method a fluorescein isothiocyanate (FITC) reagent is used to measure adducts (R) from electrophiles with a modified Edman procedure. By using LC-MS/MS in product ion scan mode, a new peak was identified and the obtained MS data indicated that this adduct could originate from methyl vinyl ketone (MVK). Incubation of human-, sheep- and bovine blood with MVK increased the signal of the identified peak. By comparing the LC-MS/MS data from the unknown background peak with data obtained from synthesized fluorescein thiohydantoin (FTH) standards of the MVK adduct to valine and d(8)-valine, the identity of this adduct was confirmed. The MVK adduct was shown present in human blood (∼35 pmol/g globin, n=3) and only just above LOD in bovine blood, n=1 (LOD=2 pmol/g globin). MVK reacts, in similarity with acrylamide, via Michael addition. MVK is known to occur in the environment and has earlier been observed in biological samples, which means that there are possible natural and anthropogenic exposure sources. Analysis of an Hb adduct from MVK in humans has to our knowledge not been described before.

Effects of benthos, temperature, and dose on the fate of hexabromocyclododecane in experimental coastal ecosystems

The authors studied the fate of the brominated flame retardant hexabromocyclododecane (HBCDD) added in a particulate suspension to experimental ecosystems assembled from brackish (Baltic Sea) coastal bays. Two experiments examined how benthic macrofauna (over 21 d) and increased temperature (14 d) affected HBCDD concentrations and fractionation of α, β, and γ diastereomers in the water, sediment, and biota. A third experiment run over 3 seasons (231 d), studied the effect of HBCDD dose on the same endpoints. In all treatments of the 3 experiments, HBCDD partitioned mainly to the sediment, and this proportion increased with time. Presence of macrofauna tended to increase the HBCDD concentration in the sediment and decreased its concentration in the water. Increased temperature (+ 5°C) decreased the amount of HBCDD in sediment and water but not in the filter- and deposit-feeding infaunal bivalves (Macoma balthica). The partitioning between water, sediment, and biota was not concentration dependent. In all treatments, sediment became enriched in γ-HBCDD, M. balthica in α-HBCDD, and water in α- and β-HBCDD. Bioaccumulation of HBCDD in M. balthica was high in all experiments (log biota-sediment accumulation factor [BSAF] > 1.25), the α diastereomer contributing the most (log BSAF 2.1-5.2). There is a risk of trophic transfer of HBCDD from benthic to pelagic food webs, as well as secondary poisoning of marine consumers.