Stroke-derived neutrophils demonstrate higher formation potential and impaired resolution of CD66b + driven neutrophil extracellular traps.

BACKGROUND: Recent evidence suggests a merging role of immunothrombosis in the formation of arterial thrombosis. Our study aims to investigate its relevance in stroke patients. METHODS: We compared the peripheral immunological profile of stroke patients vs. healthy controls. Serum samples were functionally analyzed for their formation and clearance of Neutrophil-Extracellular-Traps. The composition of retrieved thrombi has been immunologically analyzed. RESULTS: Peripheral blood of stroke patients showed significantly elevated levels of DNAse-I (p < 0.001), LDG (p = 0.003), CD4 (p = 0.005) as well as the pro-inflammatory cytokines IL-17 (p < 0.001), INF-γ (p < 0.001) and IL-22 (p < 0.001) compared to controls, reflecting a TH1/TH17 response. Increased counts of DNAse-I in sera (p = 0.045) and Neutrophil-Extracellular-Traps in thrombi (p = 0.032) have been observed in patients with onset time of symptoms longer than 4,5 h. Lower values of CD66b in thrombi were independently associated with greater improvement of NIHSS after mechanical thrombectomy (p = 0.045). Stroke-derived neutrophils show higher potential for Neutrophil-Extracellular-Traps formation after stimulation and worse resolution under DNAse-I treatment compared to neutrophils derived from healthy individuals. CONCLUSIONS: Our data provide new insight in the role of activated neutrophils and Neutrophil-Extracellular-Traps in ischemic stroke. Future larger studies are warranted to further investigate the role of immunothrombosis in the cascades of stroke. TRIAL REGISTRATION: DRKS, DRKS00013278, Registered 15 November 2017, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00013278.

Correction to: Metabolites of the alkyl pyrrolidone solvents NMP and NEP in 24-h urine samples of the German Environmental Specimen Bank from 1991 to 2014.

The article Metabolites of the alkyl pyrrolidone solvents NMP and NEP in 24-h urine samples of the German Environmental Specimen Bank from 1991 to 2014.

Metabolites of the alkyl pyrrolidone solvents NMP and NEP in 24-h urine samples of the German Environmental Specimen Bank from 1991 to 2014.

PURPOSE: The aim of this study was to get a first overview of the exposure to the solvents and reproductive toxicants N-methyl-2-pyrrolidone (NMP) and N-ethyl-2-pyrrolidone (NEP) in Germany. NMP and NEP metabolite concentrations were determined in 540 24-h urine samples of the German Environmental Specimen Bank collected from 1991 to 2014. With these data we were able to investigate NMP/NEP exposures over time and to evaluate associated risks. METHODS: NMP metabolites 5-hydroxy-N-methyl-2-pyrrolidone (5-HNMP) and 2-hydroxy-N-methylsuccinimide (2-HMSI) and NEP metabolites 5-hydroxy-N-ethyl-2-pyrrolidone (5-HNEP) and 2-hydroxy-N-ethylsuccinimide (2-HESI) were determined by stable isotope dilution analysis using solid phase extraction followed by derivatization (silylation) and GC-EI-MS/MS. RESULTS: We were able to quantify 5-HNMP and 2-HMSI in 98.0 and 99.6% and 5-HNEP and 2-HESI in 34.8 and 75.7% of the samples. Metabolite concentrations were rather steady over the timeframe investigated, even for NEP which has been introduced as an NMP substitute only in the last decade. Calculated median daily intakes in 2014 were 2.7 µg/kg bw/day for NMP and 1.1 µg/kg bw/day for NEP. For the combined risk assessment of NMP and NEP exposure, the hazard index based on the human biomonitoring assessment I values (HBM I values) was less than 0.1. CONCLUSIONS: Based on the investigated subpopulation of the German population, individual and combined NMP and NEP exposures were within acceptable ranges in the investigated timeframe. Sources of NEP exposure in the 90s and 00s remain elusive.

Quantification of N-phenyl-2-naphthylamine by gas chromatography and isotope-dilution mass spectrometry and its percutaneous absorption ex vivo under workplace conditions.

N-Phenyl-2-naphthylamine (P2NA) is an antioxidant used to protect rubbers from flex-cracking. P2NA can be converted in vivo to 2NA, one of the most potent bladder carcinogens. Here, we report the specific and ultra-sensitive quantification of P2NA in the receptor fluid of Franz diffusion cells by gas chromatography and isotope-dilution tandem-mass spectroscopy (GC-MS/MS). The experimental conditions were optimized to minimize losses of P2NA due to surface absorption on glass, plastic, and rubber material, and subsequently validated. Static and dynamic diffusion cell conditions were used to study the percutaneous penetration of P2NA into freshly prepared porcine skin. The experimental settings closely resembled those of the printing industry in the 1960s/1970s in Germany where P2NA-containing solutions in dichloromethane have been used. P2NA penetrated the skin at very low levels (0.02 ± 0.01 µg/cm2/h) with a cumulative penetrated amount of 0.80 ± 0.26 µg/cm2, a lag time of 6.33 ± 2.21 h and under dynamic conditions. Compared to the receptor fluid, 10-40-fold higher concentrations were found in the skin, predominantly in the dermis and the stratum corneum. Dichloromethane acted as a penetration enhancer by increasing the cumulative penetrated amounts and the recovery of P2NA in both the receptor fluid and the skin, while shortening its lag time. However, the flux remained unaffected. Due to its accumulation in subcutaneous layers, we finally proved that P2NA is continuously released into the receptor fluid despite exposure cessation up to 160 h. Overall, the results show that close attention has to be paid to dermal absorption of P2NA in exposed workers.

Correction to: Quantification of N-phenyl-2-naphthylamine by gas chromatography and isotope-dilution mass spectrometry and its percutaneous absorption ex vivo under workplace conditions.

The article 'Quantification of N-phenyl-2-naphthylamine by gas chromatography and isotope-dilution mass spectrometry and its percutaneous absorption ex vivo under workplace conditions' written by Heiko Udo Käfferlein, was originally published electronically on the publisher's internet portal (currently SpringerLink) on 12th September 2017 without open access.

Human metabolism and excretion kinetics of aniline after a single oral dose.

Aniline is an important source material in the chemical industry (e.g., rubber, pesticides, and pharmaceuticals). The general population is known to be ubiquitously exposed to aniline. Thus, assessment of aniline exposure is of both occupational and environmental relevance. Knowledge on human metabolism of aniline is scarce. We orally dosed four healthy male volunteers (two fast and two slow acetylators) with 5 mg isotope-labeled aniline, consecutively collected all urine samples over a period of 2 days, and investigated the renal excretion of aniline and its metabolites by LS-MS/MS and GC-MS. After enzymatic hydrolysis of glucuronide and sulfate conjugates, N-acetyl-4-aminophenol was the predominant urinary aniline metabolite representing 55.7-68.9 % of the oral dose, followed by the mercapturic acid conjugate of N-acetyl-4-aminophenol accounting for 2.5-6.1 %. Acetanilide and free aniline were found only in minor amounts accounting for 0.14-0.36 % of the dose. Overall, these four biomarkers excreted in urine over 48 h post-dose represented 62.4-72.1 % of the oral aniline dose. Elimination half-times were 3.4-4.3 h for N-acetyl-4-aminophenol, 4.1-5.5 h for the mercapturic acid conjugate, and 1.3-1.6 and 0.6-1.2 h for acetanilide and free aniline, respectively. Urinary maximum concentrations of N-acetyl-4-aminophenol were reached after about 4 h and maximum concentrations of the mercapturic acid conjugate after about 6 h, whereas concentrations of acetanilide and free aniline peaked after about 1 h. The present study is one of the first to provide reliable urinary excretion factors for aniline and its metabolites in humans after oral dosage, including data on the predominant urinary metabolite N-acetyl-4-aminophenol, also known as an analgesic under the name paracetamol/acetaminophen.

Effects of benzo[a]pyrene, aromatic amines, and a combination of both on CYP1A1 activities in RT-4 human bladder papilloma cells.

The interaction of arylamines and polycyclic aromatic hydrocarbons (PAH) is of particular interest in the etiology of bladder cancer. The aim of this study was to (1) examine the metabolic capacity of RT-4 human bladder papilloma cells and (2) investigate the influence of aromatic amines on the induction of cytochrome P-450 1A1 (CYP1A1) activity and their effects on benzo[a]pyrene (BaP)-induced CYP1A1 activities. Cells were incubated for 24 h with different concentrations of BaP, 1- or 2-naphthylamine (NA), 2-, 3-, or 4-aminobiphenyl (ABP), and binary mixtures consisting of 1 µM BaP and different concentrations of each arylamine. Changes in CYP1A1 activities were measured at concentrations with no or only low cytotoxicity and accompanied by specific protein detection. Several phase I and II enzymes relevant to metabolism of PAH and arylamines were present in RT-4 cells. Concentration-dependent elevation in CYP1A1 activities accompanied by increasing protein levels was found after treating cells with BaP and 1- or 2-NA. The majority of synergistic effects in binary mixtures were less than additive. In contrast, concentration-dependent inhibition was observed for 2-, 3-, and 4-ABP and in both the presence and absence of BaP. Our results suggest that RT-4 cells represent a reliable model cell line to study arylamine- and PAH-induced effects in vitro and that BaP-induced CYP1A1 activities are modulated by aromatic amines. In general, the direction of the effect depends upon the aromatic amine, rather than being unidirectional for aromatic amines.

Human exposure to airborne aniline and formation of methemoglobin: a contribution to occupational exposure limits.

Aniline is an important starting material in the manufacture of polyurethane-based plastic materials. Aniline-derived methemoglobinemia (Met-Hb) is well described in exposed workers although information on the dose-response association is limited. We used an experimental design to study the association between aniline in air with the formation of Met-Hb in blood and the elimination of aniline in urine. A 6-h exposure of 2 ppm aniline in 19 non-smoking volunteers resulted in a time-dependent increase in Met-Hb in blood and aniline in urine. The maximum Met-Hb level in blood (mean 1.21 ± 0.29 %, range 0.80-2.07 %) and aniline excretion in urine (mean 168.0 ± 51.8 µg/L, range 79.5-418.3 µg/L) were observed at the end of exposure, with both parameters rapidly decreasing after the end of exposure. After 24 h, the mean level of Met-Hb (0.65 ± 0.18 %) returned to the basal level observed prior to the exposure (0.72 ± 0.19 %); whereas, slightly elevated levels of aniline were still present in urine (means 17.0 ± 17.1 vs. 5.7 ± 3.8 µg/L). No differences between males and females as well as between slow and fast acetylators were found. The results obtained after 6-h exposure were also comparable to those observed in four non-smoking volunteers after 8-h exposure. Maximum levels of Met-Hb and aniline in urine were 1.57 % and 305.6 µg/L, respectively. Overall, our results contribute to the risk assessment of aniline and as a result, the protection of workers from aniline-derived adverse health effects at the workplace.

Occupational exposure to polycyclic aromatic hydrocarbons and DNA damage by industry: a nationwide study in Germany.

Exposure to polycyclic aromatic hydrocarbons (PAH) and DNA damage were analyzed in coke oven (n = 37), refractory (n = 96), graphite electrode (n = 26), and converter workers (n = 12), whereas construction workers (n = 48) served as referents. PAH exposure was assessed by personal air sampling during shift and biological monitoring in urine post shift (1-hydroxypyrene, 1-OHP and 1-, 2 + 9-, 3-, 4-hydroxyphenanthrenes, SigmaOHPHE). DNA damage was measured by 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and DNA strand breaks in blood post shift. Median 1-OHP and SigmaOHPHE were highest in converter workers (13.5 and 37.2 microg/g crea). The industrial setting contributed to the metabolite concentrations rather than the air-borne concentration alone. Other routes of uptake, probably dermal, influenced associations between air-borne concentrations and levels of PAH metabolites in urine making biomonitoring results preferred parameters to assess exposure to PAH. DNA damage in terms of 8-oxo-dGuo and DNA strand breaks was higher in exposed workers compared to referents ranking highest for graphite-electrode production. The type of industry contributed to genotoxic DNA damage and DNA damage was not unequivocally associated to PAH on the individual level most likely due to potential contributions of co-exposures.

Dose-response modeling of occupational exposure to polycyclic aromatic hydrocarbons with biomarkers of exposure and effect.

In regulatory toxicology, the dose-response relationship between occupational exposure and biomarkers is of importance in setting threshold values. We analyzed the relationships between occupational exposure to polycyclic aromatic hydrocarbons (PAH) and various biomarkers of internal exposure and DNA damage with data from 284 highly exposed male workers. Personal exposure to phenanthrene and other PAHs was measured during shift and correlated with the sum of 1-, 2+9-, 3-, and 4-hydroxyphenanthrenes in post-shift urine. PAHs and hydroxyphenanthrenes were associated with DNA damage assessed in WBC as 8-oxo-7,8-dihydro-2'-deoxyguanosine/10(6) dGuo and strand breaks by Comet assay as Olive tail moment. Hydroxyphenanthrenes correlated with phenanthrene (Spearman r(s) = 0.70; P < 0.0001). No correlations could be found between strand breaks and exposure (r(s) = 0.01, P < 0.0001 for PAHs; r(s) = -0.03, P = 0.68 for hydroxyphenanthrenes). Correlations with 8-oxo-7,8-dihydro-2'-deoxyguanosine/10(6) dGuo were weakly negative (r(s) = -0.22, P = 0.004 for PAHs) or flat (r(s) = -0.08, P = 0.31 for hydroxyphenanthrenes). Linear splines were applied to assess the relationships between the log-transformed variables. All regression models were adjusted for smoking and type of industry. For hydroxyphenanthrenes, 51.7% of the variance could be explained by phenanthrene and other predictors. Up to 0.77 microg/m(3) phenanthrene, no association could be found with hydroxyphenanthrenes. Above that point, hydroxyphenanthrenes increased by a factor of 1.47 under a doubling of phenanthrene exposure (slope, 0.56; 95% confidence interval, 0.47-0.64). Hydroxyphenanthrenes may be recommended as biomarker of occupational PAH exposure, whereas biomarkers of DNA damage in blood did not show a dose-response relation to PAH exposure.

Soluble chemokine (C-X-C motif) ligand 16 (CXCL16) in urine as a novel biomarker candidate to identify high grade and muscle invasive urothelial carcinomas.

Information on biomarkers of urothelial carcinomas (UC) for clinical decision-making is limited. Here, we newly identified and verified CXCL16 as a promising novel biomarker in urine for high grade and muscle invasive UC in a cross-sectional cohort of 308 UC patients, 126 urological hospital controls, and 50 population controls using antibody arrays and ELISA. Median CXCL16 levels in urine was significantly higher in UC patients (273.2 pg/mg creatinine) compared to hospital (148.1 pg/mg) and population controls (85.1 pg/mg) with a particular preference for high grade (460.8 pg/mg), muscle invasive (535.7 pg/mg) and primary UC (327.8 pg/mg) (all p<0.0001). Group differences were confirmed after adjusting or stratifying for potential clinical and individual characteristics, such as leukocyte counts, haematuria, age, gender, and smoking status. In contrast, CXCL16 showed less discriminating power in low grade (244.3 pg/mg), non-muscle invasive (≤pT1, 251.2 pg/mg) and recurrent UC (203.9 pg/mg). In agreement with its function in immune defence, expression of CXCL16 in tissue samples of primary UC patients (n=53) showed only a weak or no immunoreactivity compared to urological hospital controls (n=32). Expression of CXCR6, the G-protein-coupled receptor of CXCL16, remained unchanged. Our findings suggest that evading the immune defence by shedding cell-surface CXCL16 and its increased elimination in urine is a molecular feature of high grade and muscle invasive UC. Therefore, urinary CXCL16 may serve as a useful, simple and non-invasive tool to identify high-risk UC with increased risk of progression at the molecular level.

Biomarkers of polycyclic aromatic hydrocarbon exposure in European coke oven workers.

Biomonitoring is an excellent method for capturing the results of all exposures, regardless of route. Coke oven workers include certain groups that have the potential for high exposure to polycyclic aromatic hydrocarbons (PAH) and other materials. Biomarkers of exposure to these agents include PAH metabolites as markers of internal dose and carcinogen-DNA adducts as measure of effective dose. The purpose of this study was to determine the levels of these biomarkers in persons with different job duties in a modern coke oven plant. We report that the mean levels of 1-hydroxypyrene (1HP) and carcinogen DNA adducts in the exfoliated urothelial cells of coke oven workers are increased the closer a group of workers is to the ovens and highest in the top oven workers with average 1HP level of 11.6 µg/l and 22 adducts per 10(9) unadducted nucleotides. Both 1HP and carcinogen DNA adduct levels increased in supervisors, area workers, side oven workers, top and side oven workers, and top oven workers, respectively. These data are the first to demonstrate an increase in target organ genotoxicity in coke oven workers and a relationship with other biomarkers. Future studies will determine the identity of the DNA adducts, their correlation with 1HP levels and the relationship between levels in individual workers.

N-Acetyl-4-aminophenol (paracetamol), N-acetyl-2-aminophenol and acetanilide in urine samples from the general population, individuals exposed to aniline and paracetamol users.

Epidemiological studies suggest associations between the use of N-acetyl-4-aminophenol (paracetamol) during pregnancy and increased risks of reproductive disorders in the male offspring. Previously we have reported a ubiquitous urinary excretion of N-acetyl-4-aminophenol in the general population. Possible sources are (1) direct intake of paracetamol through medication, (2) paracetamol residues in the food chain and (3) environmental exposure to aniline or related substances that are metabolized into N-acetyl-4-aminophenol. In order to elucidate the origins of the excretion of N-acetyl-4-aminophenol in urine and to contribute to the understanding of paracetamol and aniline metabolism in humans we developed a rapid, turbulent-flow HPLC-MS/MS method with isotope dilution for the simultaneous quantification of N-acetyl-4-aminophenol and two other aniline related metabolites, N-acetyl-2-aminophenol and acetanilide. We applied this method to three sets of urine samples: (1) individuals with no known exposure to aniline and also no recent paracetamol medication; (2) individuals after occupational exposure to aniline but no paracetamol medication and (3) paracetamol users. We confirmed the omnipresent excretion of N-acetyl-4-aminophenol. Additionally we revealed an omnipresent excretion of N-acetyl-2-aminophenol. In contrast, acetanilide was only found after occupational exposure to aniline, not in the general population or after paracetamol use. The results lead to four preliminary conclusions: (1) other sources than aniline seem to be responsible for the major part of urinary N-acetyl-4-aminophenol in the general population; (2) acetanilide is a metabolite of aniline in man and a valuable biomarker for aniline in occupational settings; (3) aniline baseline levels in the general population measured after chemical hydrolysis do not seem to originate from acetanilide and hence not from a direct exposure to aniline itself and (4) N-acetyl-2-aminophenol does not seem to be related to aniline nor to N-acetyl-4-aminophenol in man.

Biomonitoring of N-ethyl-2-pyrrolidone in automobile varnishers.

N-alkyl-2-pyrrolidones are important organic solvents for varnishes in industry. This study investigates exposure to N-ethyl-2-pyrrolidone (NEP) in varnishing of hard plastic components in an automobile plant. Two specific biomarkers of exposure, 5-hydroxy-N-ethyl-2-pyrrolidone (5-HNEP) and 2-hydroxy-N-ethylsuccinimide (2-HESI), were analyzed in urine samples of 14 workers. For this purpose, pre-shift, post-shift and next day pre-shift urine samples were collected midweek. Twelve workers performed regular work tasks (loading, wiping and packing), whereas two workers performed special work tasks including cleaning the sprayer system with organic solvents containing N-alkyl-2-pyrrolidones. Spot urine samples of nine non-exposed persons of the same plant served as controls. Median post-shift urinary levels of workers with regular work tasks (5-HNEP: 0.15 mg/L; 2-HESI: 0.19 mg/L) were ∼5-fold higher compared to the controls (0.03 mg/L each). Continuously increasing metabolite levels, from pre-shift via post-shift to pre-shift samples of the following day, were observed in particular for the two workers with the special working tasks. Maximum levels were 31.01 mg/L (5-HNEP) and 8.45 mg/L (2-HESI). No clear trend was evident for workers with regular working tasks. In summary, we were able to show that workers can be exposed to NEP during varnishing tasks in the automobile industry.

Phthalate exposure during cold plastisol application--a human biomonitoring study.

The phthalates DEHP (Diethylhexyl phthalate), DiNP (Diisononyl phthalate) and DiDP (Diisodecyl phthalate) are constituents of plastisols. We sought to obtain first data on occupational exposures to the above phthalates by analyzing their metabolites in pre- and post-shift urine samples from 5 workers in a car manufacturing plant engaged in seam sealing with a DINP based plastisol. Pre-shift samples were collected after a work-free period of at least 2 days. As a comparison group we investigated 10 employees from the same plant. The comparison group had phthalate exposures in the range of the general German population. All plastisol workers had post shift values of DiNP and DiDP metabolites that were approx. 20-times higher, and pre-shift values that were approx. 5-10 times higher than those of the general background exposure. Post-shift values of DiNP metabolites were (median [maximum]: OH-MiNP: 117 [442] µg/L; oxo-MiNP: 44.3 [175] µg/L; carboxy-MiNP: 57.8 [286]µg/L), pre shift values were (OH-MiNP: 26 [164] µg/L; oxo-MiNP 12.9 [68.6] µg/L; carboxy-MiNP: 32.3 [103] µg/L), compared to the comparison group (OH-MiNP: 6.2 [33]µg/L; oxo-MiNP: 2.8 [16] µg/L; carboxy-MiNP: 6.5 [31] µg/L). DiDP values were generally lower. Regarding DEHP we found no significant work related exposure. The dermal exposure route might play an important role for phthalates in plastisols, with possible influences on distribution and elimination kinetics and therefore data interpretation.

Levels and predictors of airborne and internal exposure to manganese and iron among welders.

We investigated airborne and internal exposure to manganese (Mn) and iron (Fe) among welders. Personal sampling of welding fumes was carried out in 241 welders during a shift. Metals were determined by inductively coupled plasma mass spectrometry. Mn in blood (MnB) was analyzed by graphite furnace atom absorption spectrometry. Determinants of exposure levels were estimated with multiple regression models. Respirable Mn was measured with a median of 62 (inter-quartile range (IQR) 8.4-320) µg/m(3) and correlated with Fe (r=0.92, 95% CI 0.90-0.94). Inhalable Mn was measured with similar concentrations (IQR 10-340 µg/m(3)). About 70% of the variance of Mn and Fe could be explained, mainly by the welding process. Ventilation decreased exposure to Fe and Mn significantly. Median concentrations of MnB and serum ferritin (SF) were 10.30 µg/l (IQR 8.33-13.15 µg/l) and 131 µg/l (IQR 76-240 µg/l), respectively. Few welders were presented with low iron stores, and MnB and SF were not correlated (r=0.07, 95% CI -0.05 to 0.20). Regression models revealed a significant association of the parent metal with MnB and SF, but a low fraction of variance was explained by exposure-related factors. Mn is mainly respirable in welding fumes. Airborne Mn and Fe influenced MnB and SF, respectively, in welders. This indicates an effect on the biological regulation of both metals. Mn and Fe were strongly correlated, whereas MnB and SF were not, likely due to higher iron stores among welders.