Thomas Schettgen

Pyrethroid exposure of the general population —is this due to diet?

Inhabitants (1177) of a residential area in Frankfurt/Main have been investigated with respect to internal exposure to pyrethroids. Biological monitoring revealed a body burden of pyrethroids. The 95th per thousand for the urinary metabolites of pyrethroids, such as permethrin and cypermethrin, cis and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-DCCA and trans-DCCA), was determined to be 0.5 and 1.4 microg/l, respectively. 95th per thousand for cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (DBCA), a specific metabolite of deltamethrin, and 4-fluoro-3-phenoxybenzoic acid (F-PBA), a metabolite of cyfluthrin, were 0.3 and 0.27 microg/l, respectively. The metabolic pattern found for these samples points out that pyrethroids are probably ingested orally with daily diet.

New gas chromatographic-mass spectrometric method for the determination of urinary pyrethroid metabolites in environmental medicine

We have developed and validated a new, reliable and very sensitive method for the determination of the urinary metabolites of the most common pyrethroids in one analytical run. After acidic hydrolysis for the cleavage of conjugates, the analytes cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl(2)CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (trans-Cl(2)CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br(2)CA), 4-fluoro-3-phenoxybenzoic acid (F-PBA) and 3-phenoxybenzoic acid (3-PBA) were extracted from the matrix with a liquid-liquid extraction procedure using n-hexane under acidic conditions. For further clean-up, NaOH was added to the organic phase and the carboxylic acids were re-extracted into the aqueous phase. After acidification and extraction into n-hexane again, the metabolites were then derivatised to volatile esters using N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamid (MTBSTFA). Separation and detection were carried out using capillary gas chromatography with mass-selective detection (GC-MS). 2-Phenoxybenzoic acid (2-PBA) served as internal standard for the quantification of the pyrethroid metabolites. The limit of detection for all analytes was 0.05 microg/l urine. The RSD of the within-series imprecision was between 2.0 and 5.4% at a spiked concentration of 0.4 microg/l and the relative recovery was between 79.3 and 93.4%, depending on the analyte. This method was used for the analysis of urine samples of 46 persons from the general population without known exposure to pyrethroids. The metabolites cis-Cl(2)CA, trans-Cl(2)CA and 3-PBA could be found in 52, 72 and 70% of all samples with median values of 0.06, 0.11 and 0.16 microg/l, respectively. Br(2)CA and F-PBA could also be detected in 13 and 4% of the urine samples.

Determination of haemoglobin adducts of acrylamide and glycidamide in smoking and non-smoking persons of the general population

Acrylamide (AA) is a food-borne toxicant suspected to be carcinogenic to humans. It is formed in the heating process of starch-containing food. Currently, there is a great discussion about the possible human health risks connected with the dietary uptake of acrylamide. Haemoglobin adducts of acrylamide and its oxidative metabolite glycidamide are both markers of biochemical effect. However, because glycidamide has a higher carcinogenic potency than acrylamide itself, the glycidamide adduct might mirror the genotoxicity better than acrylamide adducts. In order to gain more information about the human metabolism of acrylamide, we investigated a small group of persons for the effective internal doses of acrylamide and glycidamide using haemoglobin adducts as parameters of biochemical effect. The collective was subdivided into non-smokers (n=13) and smokers (n=16) by determining the smoking-specific acrylonitrile haemoglobin adduct (N-cyanoethylvaline, CEV). The mean values for the adducts of acrylamide (N-2-carbamoylethylvaline, AAVal) and glycidamide (N-(R,S)-2-hydroxy-2-carbamoylethylvaline, GAVal) in nonsmokers was 19 pmol/g globin AAVal (range 7-31 pmol/g globin) and 17 pmol/g globin GAVal (range 9-23 pmol/g globin). For smokers mean levels of AAVal were 80 pmol/g globin (range: 25-199 pmol/g globin) and those of GAVal were 53 pmol/g globin (range: 22-119 pmol/g globin). Metabolism to glycidamide turned out to be significantly more effective in non-smokers than in the higher exposed smokers. Compared with studies in rats, the metabolic conversion of acrylamide to glycidamide as measured by haemoglobin adducts seems to occur to a similar extent in humans as in rats. Risk estimations on acrylamide based on experimental data obtained in rats obviously did not overestimate the cancer risk for the general population. Furthermore, our results might indicate that the dose-response curve for acrylamide is not linear. This would be in line with the results of animal experiments on rodents.

Hemoglobin adducts of ethylene oxide, propylene oxide, acrylonitrile and acrylamide -biomarkers in occupational and environmental medicine

In a chemical plant, ethylene oxide (EO) and propylene oxide (PO) were used for the production of surfactants for the textile industry. Within health supervision, we investigated the internal exposure of the workers using hemoglobin adducts as parameters of biochemical effects. The 95th percentile for N-2-hydroxyethylvaline (HEV) was 1280 pmol/g globin (=29.4 microg/l blood) in blood from exposed workers compared with 100 pmol/g globin (or 2.3 microg/l) in controls. N-(R,S)-2-hydroxypropylvaline (HPV) both in workers and controls was below the detection limit (80 pmol/g globin or 2 microg/l). The levels of the adducts of acrylonitrile (ACN) and acrylamide (AA) were also determined, though they were mainly accounted for by smoking and diet. Median values of N-2-cyanoethylvaline (CEV) were below 4 pmol/g globin (or 0.1 microg/l) in non-smokers (n=24) and 131 pmol/g globin (or 3.3 microg/l) in smokers (n=38). Median values of N-2-carbamoylethylvaline (AAV) were 22 pmol/g globin (or 0.6 microg/l) in non-smokers compared with 89 pmol/g globin (or 2.4 microg/l) in smokers. Correlations were found between smoking habits and adduct levels of CEV and AAV.

Evidence for increased internal exposure to lower chlorinated polychlorinated biphenyls (PCB) in pupils attending a contaminated school.

External and internal exposure to six WHO-indicator congeners of polychlorinated biphenyls (PCB 28, 52, 101, 138, 153, 180) as well as subjective health complaints of a group of 377 pupils attending a PCB-contaminated school were compared with a control group of 218 pupils attending a non-contaminated school. Indoor air of the contaminated school revealed total PCB concentrations (sum of six indicator congeners times 5) ranging between 690 and 20,800 ng/m3 (median 2044 ng/m3). The lower chlorinated congeners PCB 28, 52, 101 were the prevailing contaminants (median 33, 293, and 66 ng/m3). Using improved analytical procedures at least one of the lower chlorinated congeners could be detected in 95% of the blood samples of pupils attending the contaminated school. Median concentrations for PCB 28, 52, 101, and for the sum of lower chlorinated congeners were 6, 9, 5, and 22 ng/l blood plasma, respectively, whereas the corresponding values in the control group were all < 1 ng/l. In contrast, no significant differences were found for the higher chlorinated congeners (PCB 138, 153, 180) which were detected in 1-2 orders of magnitude higher concentrations in both groups. Due to the dietary intake of these congeners similar total PCB levels were found (95th percentile 1070 and 1010 ng/l plasma in participants of the contaminated and control school). Using the Giessen Subjective Complaint List for Children and Adolescents no statistically significant differences in health complaints were observed between both groups of children. It is concluded that exposure of pupils to PCB in indoor air of the contaminated school caused increased blood concentrations of lower chlorinated congeners. Compared to background levels the detected excess body burden was very low indicating no additional health risk. Exposure was not associated to any specific subjective complaints.

A first approach to estimate the internal exposure to acrylamide in smoking and non-smoking adults from Germany

Since the formation of acrylamide (AA) in the heating process of starch-containing food could be demonstrated and high contents of this substance were found in commercial food products, there is a great discussion about the possible human health risks connected with this dietary exposure. In order to determine the body burden of the general population in Germany caused by this uptake, we investigated the internal exposure to acrylamide and acrylonitrile in a group of 72 persons using haemoglobin adducts as parameters of biochemical effects. The collective was subdivided into non-smokers and smokers basing on the results of the smoking-specific acrylonitrile adduct (N-cyanoethylvaline, CEV). The median value for the adduct of AA (N-2-carbamoylethylvaline, AAV) in 25 non-smokers was 21 pmol/g globin (approximately 0.6 microgram/l blood) with a 95 percentile of 46 pmol/g globin (approximately 1.3 micrograms/l) (LOD: 12 pmol/g globin). The median level for AAV in smokers (n = 47) was found to be 85 pmol/g globin (approximately 2.3 micrograms/l blood) with a 95 percentile of 159 pmol/g globin (approximately 4.3 micrograms/l blood). Based on these results about 60 micrograms AA/d are taken up by adult non-smoking persons. According to calculations of WHO and US EPA this background exposure would lead to a cancer risk between 6 x 10(-4) and 3.6 x 10(-3). Our results confirm a body burden to AA even in persons from the non-smoking general population in Germany that is most probably caused by dietary uptake. Smoking habits considerably contribute to the level of this adduct.

Hemoglobin adducts and mercapturic acid excretion of acrylamide and glycidamide in one study population.

The aim of this study was to determine the relationship between the oxidative and reductive metabolic pathways of acrylamide (AA) in the nonsmoking general population. For the first time both the blood protein adducts and the urinary metabolites of AA and glycidamide (GA) were quantified in an especially designed study group with even distribution of age and gender. The hemoglobin adducts N-carbamoylethylvaline (AAVal) and N-( R, S)-2-hydroxy-2-carbamoylethylvaline (GAVal) were detected by GC-MS/MS in all blood samples with median levels of 30 and 34 pmol/g of globin, respectively. Concentrations ranged from 15 to 71 pmol/g of globin for AAVal and from 14 to 66 pmol/g of globin for GAVal. The ratio GAVal/AAVal was 0.4-2.7 (median = 1.1). The urinary metabolites were determined by LC-MS/MS. Of all urine samples examined 99% of N-acetyl- S-(2-carbamoylethyl)- l-cysteine (AAMA) levels and 73% of N-( R/ S)-acetyl- S-(2-carbamoyl-2-hydroxyethyl)- l-cysteine (GAMA) levels were above the LOD (1.5 microg/L). Concentrations ranged from <LOD to 229 microg/L (median = 29 microg/L) for AAMA and from <LOD to 85 microg/L (median = 7 microg/L) for GAMA. The ratio of GAMA/AAMA varied from 0.004 to 1.4 (median = 0.3). Using hemoglobin adduct levels in blood and mercapturic acid excretion in urine for calculation of daily AA intake gave practically identical values. The median daily intakes were 0.43 (0.21-1.04) microg/kg of body weight(bw)/day using Hb adducts and 0.51 (<LOD-2.32) microg/kg of bw/day using mercapturic acids for calculations. Children take up approximately 1.3-1.5 times more AA per kilogram of body weight than adults. The ratio GAMA/AAMA is significantly higher in the group of young children (6-10 years) with a median level of 0.5. A gender-related difference in internal exposure and metabolism was not observed.

Simultaneous quantification of haemoglobin adducts of ethylene oxide, propylene oxide, acrylonitrile, acrylamide and glycidamide in human blood by isotope-dilution GC/NCI-MS/MS.

Haemoglobin adducts are highly valuable biomarkers of cumulative exposure to carcinogenic substances. We have developed and applied an analytical method for the simultaneous quantification of five haemoglobin adducts of important occupational and environmental carcinogens. The N-terminal adducts were determined with gas chromatography as pentafluorophenylthiohydantoine derivatives according to the modified Edman-procedure and subsequent acetonization of the glycidamide adduct N-(R,S)-2-hydroxy-2-carbamoylethylvaline (GAVal). The use of self-synthesized labelled internal standards in combination with tandem mass spectrometry using negative chemical ionisation guarantees both high accuracy and sensitivity of our determination. The limit of detection for N-2-hydroxyethylvaline (HEVal), N-(R,S)-2-hydroxypropylvaline (HPVal), N-2-carbamoylethylvaline (AAVal) and N-(R,S)-2-hydroxy-2-carbamoylethylvaline (GAVal) was 2 pmol/g globin, for N-2-cyanoethylvaline (CEVal) it was determined as 0.5 pmol/g globin, which was sufficient to determine the background levels of these adducts in the non-smoking general population. The between-day-precision for all analytes using a human blood sample as quality control material ranged from 4.7 to 12.3%. We investigated blood samples of a small group (n=104) of non-smoking persons of the general population for the background levels of these haemoglobin adducts. The median values for HEVal, HPVal, CEVal, AAVal and GAVal in a group of 92 non-smoking persons were 18.1, 4.1, <0.5, 29.9 and 35.2 pmol/g globin, respectively. The adduct levels in 12 persons reporting exposure to passive smoke at home were similar for most adducts with median values of 17.2, 4.1, 1.0, 24.9 and 29.7 pmol/g globin for HEVal, HPVal, CEVal, AAVal and GAVal, respectively. Our results point to an elevated uptake of acrylonitrile caused by passive smoking as indicated by higher levels of the corresponding haemoglobin adduct CEVal.

Influence of Diet on Exposure to Acrylamide – Reflections on the Validity of a Questionnaire

Aim: This pilot study attempts to assess how far the standardized questionnaires are a valid tool to detect the food-related burden of acrylamide. Acrylamide is a toxic substance classified by the International Agency of Research on Cancer, as well as the Deutsche Forschungsgemeinschaft, as a probable human carcinogen. Methods: A venous blood sample was taken in order to determine the smoking-specific acrylnitrile adduct N-cyanoethylvaline and the acrylamide adduct N-2-carbamoylethylvaline in a female study population expecting delivery soon. A standardized questionnaire was used to determine the consumption of acrylamide-contaminated food. The results of our questionnaire were transferred to a linear evaluation system. Finally, anamnestic data of the questionnaire were correlated to objective parameters such as blood levels of hemoglobin adducts of acrylamide and acrylonitrile. Results: A positive correlation between the acrylamide intake and the levels of hemoglobin adducts in our study population was not proven. Conclusions: Evaluation of food-related exposure to acrylamide is difficult due to several reasons. Firstly, the validity of anamnestic data strongly depends on the patient’s ability to remember precisely all consumed food (quality as well as quantity) over a 3-month period. In addition, the contamination of acrylamide in food varies from one product to another; even the contamination of the same product is variable. Therefore, the missing correlation between the questionnaire and hemoglobin adduct rates is rather due to restricted validity of anamnestic data.

Use of plasma exchange or double filtration plasmapheresis to reduce body burden of polychlorinated biphenyls: A pilot trial

The aim of the study was to examine whether plasma exchange (PE) or selective double filtration plasmapheresis (DFPP) is able to reduce the internal Polychlorinated biphenyl (PCB) burden of highly exposed participants of the health effects in high-level exposure to PCB (HELPcB) cohort. HELPcB is a surveillance program for former PCB-exposed workers of a German capacitor recycling company. After comparative evaluation of PE and DFPP in a phase I, DFPP was chosen as method for further treatment. In phase II, five participants underwent DFPP at weekly intervals for the duration of 12 weeks. Six PCB species were selected as indicators and were analyzed in the plasma before and after each treatment and 4 weeks after the last treatment. The PCB levels before and after each DFPP treatment showed a significant reduction in PCB blood levels; however, there was no significant change in PCB levels within the samples collected before each treatment as compared with the samples collected in the following week before treatment. Even the difference between PCB levels at the onset of the study and 4 weeks after the last treatment was not significant. The results of this pilot trial do not encourage further investigations in using therapeutic apheresis to reduce the PCB body burden.