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Wigger T.,University of Munster | Seidel A.,Biochemical Institute for Environmental Carcinogens | Karst U.,University of Munster
Chemosphere | Year: 2017

Electrochemistry coupled to liquid chromatography and mass spectrometry was used for simulating the biological and environmental fate of polycyclic aromatic hydrocarbons (PAHs) as well as for studying the PAH degradation behavior during electrochemical remediation. Pyrene and benzo[a]pyrene were selected as model compounds and oxidized within an electrochemical thin-layer cell equipped with boron-doped diamond electrode. At potentials of 1.2 and 1.6 V vs. Pd/H2, quinones were found to be the major oxidation products for both investigated PAHs. These quinones belong to a large group of PAH derivatives referred to as oxygenated PAHs, which have gained increasing attention in recent years due to their high abundance in the environment and their significant toxicity. Separation of oxidation products allowed the identification of two pyrene quinone and three benzo[a]pyrene quinone isomers, all of which are known to be formed via photooxidation and during mammalian metabolism. The good correlation between electrochemically generated PAH quinones and those formed in natural processes was also confirmed by UV irradiation experiments and microsomal incubations. At potentials higher than 2.0 V, further degradation of the initial oxidation products was observed which highlights the capability of electrochemistry to be used as remediation technique. © 2017 Elsevier Ltd

Schumacher F.,German Institute of Human Nutrition | Florian S.,German Institute of Human Nutrition | Schnapper A.,German Institute of Human Nutrition | Schnapper A.,Hannover Medical School | And 6 more authors.
Archives of Toxicology | Year: 2014

1-Methoxy-3-indolylmethyl (1-MIM) glucosinolate, a secondary metabolite of Brassicales species, and its breakdown product 1-MIM alcohol are mutagenic in cells in culture after activation by plant β-thioglucosidase and human sulphotransferase, respectively. In the present study, we administered these compounds orally to mice to study time course, dose dependence, tissue distribution and cellular localization of the 1-MIM DNA adducts formed. We used isotope-dilution ultra-performance liquid chromatography-tandem mass spectrometry to quantify the adducts and raised an antiserum for their immunohistochemical localization. Both compounds formed the same adducts, N 2-(1-MIM)-2′-deoxyguanosine and N 6-(1-MIM)- 2′-deoxyadenosine, approximately in a 3.3:1 ratio. 1-MIM glucosinolate primarily formed these adducts in the large intestine, with a luminal-basal gradient, probably due to activation by thioglucosidase from intestinal bacteria. 1-MIM alcohol formed higher levels of adduct than the glucosinolate. Unlike after treatment with the glucosinolate, luminal and basal enterocytes were similarly affected in caecum, and liver and stomach were additional important target tissues. Maximal adduct levels were reached 8 h after the administration of both compounds. The hepatic DNA adducts persisted for the entire observation period (48 h), whereas those in large intestine rapidly declined due to cell turnover, as verified by immunohistochemistry. Hepatic adduct formation was focused on the periportal hepatocytes with concomitant depletion of glycogen, p53 activation and p21 induction. Adduct formation in caecum was associated with massive apoptosis, p53 activation and p21 induction, in particular after treatment with 1-MIM alcohol. It remains to be studied whether similar effects occur in humans after the consumption of Brassicales species. © 2013 Springer-Verlag Berlin Heidelberg.

Monien B.H.,German Institute of Human Nutrition | Engst W.,German Institute of Human Nutrition | Barknowitz G.,German Institute of Human Nutrition | Seidel A.,Biochemical Institute for Environmental Carcinogens | Glatt H.,German Institute of Human Nutrition
Chemical Research in Toxicology | Year: 2012

5-Hydroxymethylfurfural (HMF), a heterocyclic product of the Maillard reaction, is a ubiquitous food contaminant. It has demonstrated hepatocarcinogenic activity in female mice. This effect may originate from sulfo conjugation of the benzylic alcohol yielding 5-sulfooxymethylfurfural (SMF), which is prone to react with DNA via nucleophilic substitution. Indeed, we showed that HMF induces gene mutations in Chinese hamster V79 cells engineered for the expression of human (h) sulfotransferase (SULT)1A1 but not in parental V79 cells. In order to identify potential DNA adducts, we incubated DNA samples with SMF or HMF in aqueous solution. Modified DNA was digested and surveyed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for adducts that may be formed by nucleosides either via nucleophilic substitution at the electrophilic carbon atom of SMF or via imine formation with the aldehyde group present in HMF and SMF. The most abundant adducts formed from SMF, N 6-((2-formylfuran-5-yl)methyl)-2′-deoxyadenosine (N 6-FFM-dAdo) and N2-((2-formylfuran-5-yl)methyl)-2′- deoxyguanosine (N2-FFM-dGuo), were synthesized, purified, and characterized by 1H NMR. Imine adducts were only detected when DNA was incubated with very high levels of HMF following reduction of the imines to corresponding secondary amines by NaBH3CN. Sensitive techniques based on LC-MS/MS multiple reaction monitoring for the quantification of the adducts in DNA samples were devised using isotope-labeled [15N 5]N6-FFM-dAdo and [13C10, 15N5]N2-FFM-dGuo as internal standards. Both 5-methylfurfuryl adducts were detected in DNA from V79-hSULT1A1 treated with HMF but not in DNA from V79 control cells. Considering the lack of other known mutagenic metabolites, we hypothesize that the hepatocarcinogenic potential of HMF originates from the formation of mutagenic SMF. © 2012 American Chemical Society.

Schumacher F.,German Institute of Human Nutrition | Engst W.,German Institute of Human Nutrition | Monien B.H.,German Institute of Human Nutrition | Florian S.,German Institute of Human Nutrition | And 6 more authors.
Analytical Chemistry | Year: 2012

1-Methoxy-3-indolylmethyl (1-MIM) glucosinolate, present at substantial levels in several food crops (e.g., broccoli and cabbage), forms DNA adducts in vitro and is mutagenic to bacterial and mammalian cells after activation by the plant enzyme myrosinase. Moreover, a breakdown product, 1-MIM alcohol, is metabolized to a secondary reactive intermediate by some mammalian sulfotransferases (SULTs). First, we incubated herring-sperm DNA with 1-MIM glucosinolate in the presence of myrosinase. We identified and synthesized the predominant adducts, N2-(1-MIM)-dG and N6-(1-MIM)-dA, and developed an UPLC-ESI-MS/MS method for their specific detection using isotopic dilution. Second, we demonstrated both DNA adducts in target cells (Salmonella typhimurium TA100 and Chinese hamster V79) of standard mutagenicity tests treated with 1-MIM glucosinolate/myrosinase as well as in 1-MIM alcohol-treated Salmonella and V79 cells engineered for expression of human SULT1A1. Similar excesses of N2-(1-MIM)-dG over N6-(1-MIM)-dA adducts were found in all cellular models independent of the test compound (1-MIM glucosinolate or alcohol), whereas dA adducts predominated in the cell-free system. Finally, we detected both DNA adducts in colon tissue of a mouse orally treated with 1-MIM glucosinolate. We are going to use this specific and sensitive method for investigating genotoxic risks of food-borne exposure to 1-MIM glucosinolate in animal and human studies. © 2012 American Chemical Society.

PubMed | Biochemical Institute for Environmental Carcinogens and German Institute of Human Nutrition
Type: Comparative Study | Journal: Archives of toxicology | Year: 2016

5-Hydroxymethylfurfural (HMF) and furfuryl alcohol (FFA) are moderately potent rodent carcinogens that are present in thermally processed foodstuffs. The carcinogenic effects were hypothesized to originate from sulfotransferase (SULT)-mediated bioactivation yielding DNA-reactive and mutagenic sulfate esters, a confirmed metabolic pathway of HMF and FFA in mice. It is known that orthologous SULT forms substantially differ in substrate specificity and tissue distribution. This could influence HMF- and FFA-induced carcinogenic effects. Here, we studied HMF and FFA sulfoconjugation by 30 individual SULT forms of humans, mice and rats. The catalytic efficiencies (k cat/K M) of HMF sulfoconjugation of human SULT1A1 (13.7s(-1)M(-1)), mouse Sult1a1 (15.8s(-1)M(-1)) and 1d1 (4.8s(-1)M(-1)) and rat Sult1a1 (5.3s(-1)M(-1)) were considerably higher than those of all other SULT forms investigated (0.73s(-1)M(-1)). FFA sulfoconjugation was monitored using adenosine as a nucleophilic scavenger for the reactive 2-sulfoxymethylfuran (t 1/2=20s at 37C). The resulting adduct N (6)-((furan-2-yl)methyl)-adenosine (N (6)-MF-A) was quantified by isotope-dilution UPLC-MS/MS. The rates of N (6)-MF-A formation showed that hSULT1A1 and its orthologues in mice and rats were also the most important contributors to FFA sulfoconjugation in each of the species. Taken together, the catalytic capacity of hSULT1A1 is comparable to that of mSult1a1 in mice, the species in which carcinogenic effects of HMF and FFA were detected. This is of primary concern due to the expression of hSULT1A1 in many different tissues.

Gminski R.,Albert Ludwigs University of Freiburg | Decker K.,Albert Ludwigs University of Freiburg | Heinz C.,Albert Ludwigs University of Freiburg | Seidel A.,Biochemical Institute for Environmental Carcinogens | And 6 more authors.
Environmental and Molecular Mutagenesis | Year: 2011

Until now, the adverse effects of toner powders on humans have been considered to be minimal. However, several recent reports have suggested possible significant adverse health effects from toner dust inhalation. The aim of this study was to evaluate the genotoxic potential of black toner powders in vitro. For the study of DNA damage, A549 cells were exposed to toner-powder suspensions and to their DMSO extracts, and then subjected to the comet assay and to the in-vitro cytokinesis block micronucleus test (CB-MNvit). Cytotoxic effects of the toner samples were assessed by the erythrosin B assay. Furthermore, size, shape, and composition of the toner powders were investigated. None of the three toner powders or their DMSO extracts reduced cell viability; however, they did induce DNA damage and formed micronuclei at concentrations from 80 to 400 μg cm -2, although to a varying extent. All toner powders contain considerable amounts of the pigments carbon black and magnetite (Fe 3O 4) as well as small amounts of polycyclic aromatic hydrocarbons (PAHs). The overall results of our in-vitro study suggest that the investigated toner-powder samples are not cytotoxic but genotoxic. From the results of the physical and chemical characterization, we conclude that metals and metalloids as components of magnetite, or PAHs as components of the carbon-bearing material, are responsible for the genotoxic effects. Further research is necessary to determine the relevance of these in-vitro observations for private and occupational toner powder exposure. Environ. Mol. Mutagen., 2011. © 2010 Wiley-Liss, Inc.

Lagerqvist A.,University of Stockholm | Hakansson D.,University of Stockholm | Lundin C.,Karolinska Institutet | Prochazka G.,Karolinska Institutet | And 8 more authors.
DNA Repair | Year: 2011

Polycyclic aromatic hydrocarbons (PAH) are an important class of environmental contaminants many of which require metabolic activation to DNA-reactive bay or fjord region diolepoxides (DE) in order to exert their mutagenic and carcinogenic effects. In this study, the mutagenicity of the bay region diolepoxides (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and (±)-anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrodibenzo[a,h]anthracene (DBADE) and the fjord region diolepoxides (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]-pyrene (DBPDE) and (±)-anti-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]-phenanthrene (BPhDE) was compared in nucleotide excision repair (NER) proficient and deficient hamster cell lines. The 32P-postlabelling assay was applied to analyze DNA adduct levels and the Hprt gene mutation assay for monitoring mutations. Previously, we found that the mutagenicity per adduct was four times higher for DBPDE compared to BPDE in NER proficient cells. In these same cells, the mutagenicity of DBADE and BPhDE adducts was now found to be significantly lower compared to that of BPDE. In NER deficient cells the highest mutagenicity per adduct was found for BPDE and there was a tenfold and fivefold difference when comparing the BPDE data with the DBADE and BPhDE data, respectively. In order to investigate to what extent the mutagenicity of the different adducts in NER proficient cells was influenced by repair or replication bypass, we measured the overall NER incision rate, the rate of adduct removal, the rate of replication bypass and the frequency of induced recombination in the Hprt gene. Since NER turned out to be an important pathway for the yield of mutations, we further analyzed the role of transcription coupled NER versus global genome NER. However, our data demonstrate that neither of these pathways seems to be the sole factor determining the mutation frequency of the four PAH-DE and that the differences in the repair efficiency of these compounds could not be related to the presence of a bay or fjord region in the parent PAH. © 2011 Elsevier B.V.

Hessel S.,German Federal Institute for Risk Assessment | John A.,Biochemical Institute for Environmental Carcinogens | Seidel A.,Biochemical Institute for Environmental Carcinogens | Lampen A.,German Federal Institute for Risk Assessment
Archives of Toxicology | Year: 2013

A wide variety of contaminants are ingested through food, among them the pro-carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) that is resorbed and partially metabolized in the enterocytes of the small intestine. Previous in vitro studies have revealed that BP phenols are excreted as Phase II metabolites including glucuronides and sulfates. This export is mediated by the breast cancer resistance protein (ABCG2). The ultimate carcinogenic Phase I BP metabolite anti-BP-7,8-dihydrodiol-9,10-epoxide (BPDE) can be detoxified by glutathione conjugate formation catalyzed by glutathione S-transferases. In the present study, differentiated human intestinal Caco-2 cells were used as a model for the human small intestine to investigate the detoxification of BPDE and excretion of stereoisomeric glutathione conjugates in the presence of an inhibitor of the glutathione-cleaving enzyme γ-glutamyl transpeptidase at the cell surface. The results indicate that the glutathione conjugates of BPDE are formed and excreted mainly to the apical and to a minor extent to the basolateral side of polarized Caco-2 monolayers. Inhibition studies revealed that the multidrug resistance-associated proteins (ABCCs) are involved in the transport of BPDE glutathione conjugates. Stable ABCC1, ABCC2 and ABCC3 knockdown cell lines were generated, thus making it possible to demonstrate that ABCC1 mediates the basolateral and ABCC2 the apical excretion of BPDE glutathione conjugates. In conclusion, the ultimate carcinogen BPDE is detoxified via glutathione conjugation and subsequently excreted by Caco-2 cells in both apical and basolateral directions. This finding is equivalent to a transport into feces as well as blood system in the in vivo situation. © 2012 Springer-Verlag.

Polanska K.,University of Lodz | Hanke W.,University of Lodz | Dettbarn G.,Biochemical Institute for Environmental Carcinogens | Sobala W.,University of Lodz | And 3 more authors.
Science of the Total Environment | Year: 2014

The aim of this study was to characterize the PAH exposure level among the non-smoking Polish pregnant women and to identify the minimal set of PAH metabolites that specifically reflect environmental PAH exposure. The study population consisted of 210 non-smoking pregnant women. The urine sample was used for analysis of the following PAH metabolites: 1-, 2-, 3-, 4-, 9-hydroxyphenanthrene (1-, 2-, 3-, 4-, 9-OH-PHE), 1-hydroxypyrene (1-OH-PYR), 1,6. +. 1,8-dihydroxypyrene (DI-OH-PYR), phenanthrene trans-1,2-dihydrodiol (PHE-1,2-diol) and phenanthrene trans-9,10-dihydrodiol (PHE-9,10-diol). The analysis of all the biomarkers was performed by gas chromatography-mass spectrometry after their derivatization. The mean PAH metabolite concentrations were in the range of 0.15 (±. 0.2) μg/g creatinine for 9-OH-PHE to 5.9 (±. 10.6) μg/g creatinine for PHE-9,10-diol. Women living in the city center had higher concentrations of 1-OH-PHE (β. = 0.6; p. = 0.04), 3-OH-PHE (β. = 0.8; p. = 0.02), 9-OH-PHE (β. = 0.9; p. = 0.02), and DI-OH-PYR (β. = 1.0; p. = 0.006) than those living outside the city center. The usage of coal for residential heating was a significant predictor of all PAH metabolites except for 9-OH-PHE (p. = 0.1) and PHE-9,10-diol (p. = 0.08). With the increasing cotinine levels we observed a significant increase in the concentrations of the following PAH metabolites: 3-OH-PHE (β. = 0.2; p. = 0.007), 4-OH-PHE (β. = 0.3; p. = 0.002), PHE-1,2-diol (β. = 0.3; p. <. 0.001), 1-OH-PYR (β. = 0.2; p. = 0.01). High-density housing, usage of coal for residential heating, cotinine level in saliva, season of urine collection and distance from the place of residence to the main road explained 26% of the variance of 3-OH-PHE and 21% of the variance of 1-OH-PHE. 2-OH-PHE, 3-OH-PHE, 9-OH-PHE and PHE-9,10-diol are sufficient to predict environmental PAH exposure. The urinary PAH biomarker levels found in this study indicate that non-smoking Polish pregnant women suffer from a higher PAH exposure than those in other western countries. This higher PAH exposure level probably poses a significant health risk for the newborns and young children and will require further attention in the future. © 2014 Elsevier B.V.

Lagerqvist A.,University of Stockholm | Hakansson D.,University of Stockholm | Frank H.,Biochemical Institute for Environmental Carcinogens | Seidel A.,Biochemical Institute for Environmental Carcinogens | Jenssen D.,University of Stockholm
Food and Chemical Toxicology | Year: 2011

Chinese hamster V79 cells were used to investigate the protective effect of four known antimutagens present in food, chlorophyllin (CHL), ellagic acid (EA), epigallocathechingallate (EGCG) and benzylisothiocyanate (BITC), against potent mutagenic polycyclic aromatic hydrocarbon diol epoxides (PAH-DE) derived from benzo[. a]pyrene (BP), dibenzo[. a,h]anthracene (DBA), dibenzo[. a,l]pyrene (DBP), and benzo[. c]phenanthrene (BPh) known to be deposited on crops from polluted ambient air or formed during food processing. As fjord-region PAH-DE are more toxic and mutagenic than bay-region PAH-DE, we adjusted the concentrations of PAH-DE to induce approximately the same levels of adducts. The studies were performed using an assay indicating toxicity in terms of reduced cell proliferation together with the V79 Hprt assay for monitoring mutant frequencies. CHL significantly increased the survival and showed a protective effect against the mutagenicity of all PAH-DE. A significant protective effect of EA was found towards the mutagenicity of BPDE, DBPDE and BPhDE and with EGCG for BPDE and BPhDE. BITC had a slight positive effect on the mutagenicity of DBADE and BPhDE. Taken together, a novel and unexpected finding was that the antimutagenic activity could differ as much as by a factor of 7 towards four carcinogenic PAH metabolites being relatively similar in structure and genotoxic activity. © 2010 Elsevier Ltd.

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