Ultra-high sensitive analysis of 3-hydroxybenzo[a]pyrene in human urine using GC-APLI-MS.

Urinary 3-hydroxybenzo[a]pyrene (3-OH-BaP) is a known biomarker for human exposure to carcinogenic polycyclic aromatic hydrocarbons (PAH). In this work, a new method for the ultra-sensitive quantification of this biomarker has been developed using the hyphenation of gas chromatography and atmospheric pressure laser ionization-mass spectrometry (GC-APLI-MS). In combination with an advanced sample preparation, a limit of detection (LOD) of 0.6 pg/L was achieved which is an improvement by a factor of at least 28 compared with existing methods. The limit of quantification (LOQ) is 1.8 pg/L. With this set-up 3-OH-BaP could be analyzed in urine samples of 7 smokers and 7 non-smokers. Concentrations ranged from 37 to 270 pg/L for non-smokers and from 374 to 1171 pg/L for smokers. For the first time, 3-OH-BaP was quantifiable in all non-smoker samples as no value was below the LOQ. Correlation of the urinary 3-OH-BaP values with the number of daily smoked cigarettes and with urinary cotinine values shows a clear relationship between 3-OH-BaP content and smoking habits. This innovative analytical method enables monitoring of low levels of the biomarker 3-OH-BaP in urine of non-occupationally exposed individuals including smokers, the general population with background PAH exposure and cohorts of low exposition such as newborns and children.

GC-APLI-MS as a powerful tool for the analysis of BaP-tetraol in human urine.

For the first time gas chromatography (GC) coupled to atmospheric pressure laser ionization-mass spectrometry (APLI-MS) has been applied to the analysis of trans-anti-benzo[a]pyrene-tetraol (BaP-tetraol) formed from anti-benzo[a]pyrene diolepoxide (BPDE), the ultimate carcinogen of benzo[a]pyrene. This tetraol is considered to be an ideal urinary biomarker for polycyclic aromatic hydrocarbon (PAH) exposure as it reflects internal body burden and potentially adverse health effects. Optimization of the derivatization and the instrumental set-up led to an instrumental LOD of 0.5 fg, an improvement of the lowest instrumental LOD reported in literature of 6.4 fg by a factor of 10. The optimized procedure includes derivatization of hydroxyl groups using methyl iodide and cool on-column injection to prevent degradation of the analyte. First measurements of urine samples demonstrate that the method is capable of detecting BaP-tetraol in human urine collected from both smokers and non-smokers. Although results of analysis indicate a certain underestimation compared with literature data, this method can be expected to serve as an excellent method for the analysis of the biomarker BaP-tetraol in the future if an adequate internal standard such as 13C-labeled BaP-tetraol is applied.

Metabolites of the PAH diol epoxide pathway and other urinary biomarkers of phenanthrene and pyrene in workers with and without exposure to bitumen fumes.

PURPOSE: This study investigates the diol epoxide pathway of phenanthrene (PHE) together with phenolic metabolites of PHE and pyrene (PYR) in workers with and without exposure to bitumen fumes. METHODS: The metabolite concentrations were determined in urine samples collected from 91 mastic asphalt workers and 42 construction workers as reference group before and after shift. During shift, vapours and aerosols of bitumen were measured according to a German protocol in the workers' breathing zone. RESULTS: The median concentration of vapours and aerosols of bitumen in mastic asphalt workers was 6.3 mg/m3. Metabolite concentrations were highest in post-shift urines of smokers with bitumen exposure and showed an increase during shift. The Spearman correlations between the creatinine-adjusted concentrations of metabolites and vapours and aerosols of bitumen in non-smokers were weak (e.g. sum of Di-OH-PYR: 0.28) or negligible (e.g. 1,2-PHE-diol: 0.08; PHE-tetrol: 0.12). Metabolites from the diol epoxide pathway of PHE were excreted in higher concentrations than phenolic metabolites (post-shift, non-smoking asphalt workers: 1,2-PHE-diol 2.59 µg/g crea vs. sum of all OH-PHE 1.87 µg/g crea). 1,2-PHE-diol was weakly correlated with PHE-tetrol (Spearman coefficient 0.30), an endpoint of the diol epoxide pathway. By contrast, we found a close correlation between the sum of 1,6-DiOH-PYR and 1,8-DiOH-PYR with 1-OH-PYR (Spearman coefficient 0.76). CONCLUSIONS: Most urinary PAH metabolites were higher after shift in bitumen-exposed workers, although the association with bitumen was weak or negligible likely due to the small PAH content. The additional metabolites of PHE and PYR complete the picture of the complex metabolic pathways. Nevertheless, none of the PAH metabolites can be considered to be a specific biomarker for bitumen exposure.

Urinary toxicokinetics of di-(isononyl)-cyclohexane-1,2-dicarboxylate (DINCH(®)) in humans following single oral administration.

Phthalates such as di-2-ethylhexyl phthalate (DEHP) were restricted due to their toxic mainly reprotoxic effects. Therefore compounds such as di-(isononyl)-cyclohexane-1,2-dicarboxylate (DINCH(®)) substitute these phthalates and the exposure of humanes to substitutes may occur. Here, kinetic data are presented to assess the exposure of humans. Male and female volunteers excreted nearly the complete orally administered dose (1mg/kg b.w. corresponding to the tolerable daily intake of EFSA) of di-(isononyl)-cyclohexane-1,2-dicarboxylate within 70 h. More than 75% were excreted within 24h. Besides the main metabolite cyclohexane-1,2-dicarboxylic acid (CHDA) quantitated after hydrolysis four further metabolites of DINCH(®) are determined. Cyclohexane-1,2-dicarboxylic acid-mono-(7-hydroxy-4-methyl)octyl ester (OH-MINCH) is the main secondary metabolite with about 14% of the administered dose. Differences in excretion of all metabolites between male and females are small. Based on the generated toxicokinetic data exposure of 20 humans is recalculated from their spot urine sample collected in 2014 and the exposure are clearly below the current tolerable daily intake of 1mg/kg b.w.

Effect of prenatal polycyclic aromatic hydrocarbons exposure on birth outcomes: the Polish mother and child cohort study.

The aim of this study was to assess the impact of PAH exposure on various anthropometric measures of birth outcomes. The study population consisted of 210 nonsmoking pregnant women. Urine samples collected between 20th and 24th week of pregnancy were used for analysis of the following PAH metabolites: 1-, 2-, 3-, 4-, and 9-hydroxyphenanthrene (1-, 2-, 3-, 4-, and 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) by gas chromatography-mass spectrometry. Environmental tobacco smoke exposure (ETS) was assessed by cotinine level in saliva using a stable isotope dilution LC-ESI-MS/MS method. The mean PAH metabolite concentrations were in the range of 0.15 µg/g creatinine for 9-OH-PHE to 5.9 µg/g creatinine for PHE-9,10-diol. It was shown that none of the individual PAH exposure markers demonstrate a statistically significant influence on birth outcomes. Interestingly a statistically significant association was found between the sum of OH-PHE along with cotinine level and the cephalization index after adjusting for potential confounders (P = 0.04). This study provides evidence that combined exposure of pregnant women to common environmental pollutants such as PAH and ETS might adversely affect fetal development. Thus, reduction of human exposure to these mixtures of hazardous compounds would in particular result in substantial health benefits for newborns.

The determination of polycyclic aromatic hydrocarbons in the urine of non-smoking Polish pregnant women.

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 (ß=0.6; p=0.04), 3-OH-PHE (ß=0.8; p=0.02), 9-OH-PHE (ß=0.9; p=0.02), and DI-OH-PYR (ß=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 (ß=0.2; p=0.007), 4-OH-PHE (ß=0.3; p=0.002), PHE-1,2-diol (ß=0.3; p<0.001), 1-OH-PYR (ß=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.

Exposure to polycyclic aromatic hydrocarbons in women from Poland, Serbia and Italy--relation between PAH metabolite excretion, DNA damage, diet and genotype (the EU DIEPHY project).

Exposure of the general population to polycyclic aromatic hydrocarbons (PAH) is ubiquitous. The aim of this study was to analyze biomarkers associated with the uptake of PAH in 428 non-smoking women from Lodz (Poland), Viterbo (Italy), Belgrade (Serbia) and from the Pancevo area, where the petrochemical complex was destroyed by the air raids in 1999. Urinary excretion of PAH metabolites was lowest in Italian women, intermediary for Serbian and highest in Polish women, who predominantly excreted hydroxy phenanthrenes as metabolites of phenanthrene. Bulky DNA adduct levels were highest in Italian and Polish women. Genotype or PAH ambient air levels could not explain the dissimilarities between the study groups with respect to biomarker patterns, which probably reflected differences in life style-associated factors.

Mainstream smoke of the waterpipe: does this environmental matrix reveal as significant source of toxic compounds?

In recent years the number of waterpipe smokers has increased substantially worldwide. Here we report on the concentrations of tobacco-specific nitrosamines (TSNAs) and polycyclic aromatic hydrocarbons (PAHs) in waterpipe smoke and the analysis of selected biomarkers indicative for the body burden in waterpipe users. We further identify high amounts of unburned humectants (glycerol and propylene glycol) in the waterpipe smoke as main part of the so-called "tar" fraction. These results give cause for serious concern. For standardization we applied a machine smoking protocol. Smoke was collected on glass fiber filters and analyzed for nicotine, water, humectants, TSNAs, and PAHs. In addition, we determined carbon monoxide and found high amounts in the smoke being causative for high levels of carboxyhemoglobin (COHb) in the blood of smokers. In comparison to the reference cigarette 3R4F, the nicotine contents were 10-times higher, but TSNA levels were found lower in waterpipe smoke. This finding explained the low levels of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol detected in the urine of waterpipe smokers. Finally, the levels of benzo[a]pyrene were three times higher in waterpipe smoke compared to the reference cigarette. Altogether, the data presented in this study point to the health hazards associated with the consumption of waterpipes.

New biomarkers of occupational exposure to polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAH) are metabolized in a complex manner. Although biological activity is associated with diol-epoxide formation, phenolic metabolites have predominantly been used in human biomonitoring. In this study monohydroxylated and new metabolites were characterized as biomarkers for occupational PAH exposure. In 97 male workers, personal exposure to 16 airborne PAH compounds was measured during shift. In postshift urine, 1-hydroxypyrene and 1,6- and 1,8-dihydroxypyrene (1-OHP, DiOHP) were determined as metabolites of pyrene (P), and the sum of 1-, 2-, 3-, 4-, and 9-hydroxyphenanthrenes (OHPHE), and PHE-dihydrodiols (PHED) as metabolites of phenanthrene (PHE). The referent group comprised 21 nonsmoking construction workers. Median (interquartile range) shift concentrations of airborne P and PHE were 1.46 (0.62-4.05 microg/m(3)) and 10.9 (3.69-23.77 microg/m(3)), respectively. The corresponding parameters were 3.86 (2.08-7.44) microg/g creatinine (crn) for 1-OHP, 0.66 (0.17-1.65) microg/g crn for DiOHP, 11.44 (5.21-34.76) microg/g crn for OHPHE, and 12.28 (3.3-97.76) microg/g crn for PHED in PAH-exposed workers. The median levels of 1-OHP and OHPHE were 0.09 (0.08-0.17 microg/m(3)) and 0.59 (0.45-1.39 microg/m(3)), respectively, in the referents. PHE correlated significantly with OHPHE and PHED, and P with 1-OHP but not with DiOHP. Under a doubling of PHE, OHPHE increased by a factor of 1.56 and PHED by 1.57. With a doubling of P, 1-OHP rose by 1.31 and DiOHP by 1.27. P is predominantly metabolized into 1-OHP, whereas PHE is metabolized equally into OHPHE and PHED. Thus metabolites of PHE were found as reliable biomarkers for PAH exposure.