Wolfgang Völkel

Perfluorinated compounds : Exposure assessment for the general population in western countries

Perfluorinated compounds (PFCs) can currently be detected in many environmental media and biota, as well as in humans. Because of their persistence and their potential to accumulate they are of toxicological concern. The present review presents the current knowledge of PFC monitoring data in environmental media relevant for human exposure. In this context, PFC concentrations in indoor and ambient air, house dust, drinking water and food are outlined. Furthermore, we summarize human biomonitoring data of PFC levels in blood, breast milk, and human tissues. An estimate of the overall exposure of the general adult population is provided and compared with tolerable intake values. Using a simplified model, the average (and upper) level of daily exposure including all potential routes amounts to 1.6 ng/kg(body weight) (8.8 ng/kg(body weight)) for PFOS and 2.9 ng/kg(body weight) (12.6 ng/kg(body weight)) for PFOA in adults in the general population. The majority of exposure can be attributed to the oral route, mainly to diet. Overall, the contribution of PFOS and PFOA precursors to total exposure seems to be limited. Besides this background exposure of the general population, a specific additional exposure may occur which causes an increased PFC body burden. This has been observed in populations living near PFC production facilities or in areas with environmental contamination of PFCs. The consumption of highly contaminated fish products may also cause an increase in PFC body burdens.

QUANTITATION OF BISPHENOL A AND BISPHENOL A GLUCURONIDE IN BIOLOGICAL SAMPLES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

Bisphenol A (BPA) is a weak estrogen. Pharmacokinetic studies of BPA have demonstrated a rapid and extensive metabolism of BPA to the nonestrogenic BPA-monoglucuronide (BPA-gluc). Some investigators have reported that BPA was found at parts per billion concentrations in the tissues or urine of humans without known exposure to BPA. This work developed a rapid and sensitive method for the determination of BPA and BPA-gluc in plasma and urine based on liquid chromatography-tandem mass spectrometry. The liquid chromatography-electrospray ionization-tandem mass spectrometry method for quantitation of BPA and BPA-gluc uses stable isotope-labeled internal standards. A linear ion trap mass spectrometer permits identification and quantitation of BPA-gluc and BPA without sample workup. Development of separation conditions reduced the BPA-background in solvent samples to below 2.5 pmol/ml for BPA. Limit of quantitation (LOQ) for BPA in control urine was 15 pmol/ml; LOQ for BPA-gluc was 65 pmol/ml. Application of the method to urine samples from human subjects (n = 6) after administration of 25 μg of BPA/person (estimated maximum human daily intake) permitted the determination of excretion kinetics for BPA-gluc; BPA was below the LOD in all except two of the samples. In urine or blood samples of human subjects (n = 19) without intentional exposure to BPA, BPA concentrations were always below the limit of detection (≈2.5 pmol/ml) with or without prior glucuronidase treatment. The results show that care is required for analysis of BPA and its major metabolite BPA-gluc. The LOD obtained and the absence of detectable levels of BPA in samples from individuals suggests that general exposure of humans to BPA is much lower than the worst-case exposure scenario developed.

Pre- and Postnatal Exposure to Perfluorinated Compounds (PFCs)

Perfluorinated compounds (PFCs) are a group of chemicals widely used for many applications. In this study PFCs were investigated in maternal blood during pregnancy (at two time points) (n = 40 and 38) and 6 months after delivery (n = 47), in cord blood (n = 33) and in blood of infants six (n = 40) and nineteen months (n = 24) after birth, and monthly in breast milk samples in Germany. Concentrations in maternal serum ranged from 0.5 to 9.4 μg/L for perfluorooctane sulfonate (PFOS) and 0.7 to 8.7 μg/L for perfluorooctanoic acid (PFOA). In cord serum, the values ranged from 0.3 to 2.8 μg/L and from 0.5 to 4.2 μg/L for PFOS and PFOA, respectively. The median results from serum at six and nineteen months of age were 3.0 and 1.9 μg/L for PFOS and 6.9 and 4.6 μg/L for PFOA, respectively. In breast milk samples, PFOS ranged from <0.03 to 0.11 μg/L (median: 0.04 μg/L), while PFOA was detected only in some samples as were all other PFCs. Overall, we found low levels of PFCs in cord sera and an increase in concentrations through the first months of infant life. Although the concentrations in breast milk were low, this intake led to a body burden at the age of six months similar to (PFOS) or higher than (PFOA) that found in adults.

Determination of free and total bisphenol A in human urine to assess daily uptake as a basis for a valid risk assessment

Bisphenol A (BPA) is widely distributed and exhibits weak estrogenic activity. In contrast to BPA, the corresponding glucuronide metabolite is not estrogenic. Therefore, free and total BPA were determined in human urine samples to assess the significance of free BPA for risk assessment. In only 10% of 474 samples from 287 subjects was free BPA detected in a range from <LOD (0.3 microg/l) to 2.5 microg/l. Due to sample contamination with low amounts ( approximately 1 microg/l) of BPA, house dust samples were independently collected in homes but not from persons who provide urine samples and analysed for BPA to check for potential sources of contamination. BPA was found in the range from 117 to 1 486 microg/kg (median: 553 microg/kg) dust. Additionally, BPA and d(16)-BPA were administered to a volunteer to demonstrate the problem of contamination. In comparison to low levels of free BPA (<1.25 microg/l), no free d(16)-BPA was observed. Therefore, the maximal concentration of 2.5 microg/l observed here may be considered as an exaggerated value as a result of contamination. Nevertheless, for risk assessment a concentration of 2.5 microg/l was used and this value is 800-fold below the tolerable daily intake (TDI) of 50 microg/kg. Total BPA was determined in 147 urine samples with concentrations between <LOD and 9.3 microg/l.

Brominated flame retardants - Exposure and risk assessment for the general population.

Brominated flame retardants (BFRs) are a large group of different substances used in numerous products to prevent fire hazards. Some of them are persistent in the environment, accumulate in the food chain and are of toxicological concern, while for others current data are limited. Meanwhile, BFRs have been found in many environmental media, foods, and biota including humans. This review presents recent findings obtained from monitoring data in environmental media relevant for human exposure, as well as dietary exposure. In this context, concentrations in indoor and ambient air and in house dust are outlined. Furthermore, we summarize human biomonitoring data on BFR levels in blood and breast milk. Current estimates of the overall exposure of the general population using different relevant subsets are also addressed. All of these data are discussed in relation to currently available toxicological reference values used for risk assessment purposes. Obviously, the exposure of the general population varies considerably in different parts of the world and even within countries. Polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD) show the highest intake during infancy. While the highest intake for BDE 47 for all groups was observed in the US, the total BDE 209 and HBCD intake was highest in the UK. For HBCD and all PBDEs except BDE 209, diet accounts for a large proportion of the total intake during infancy in all countries. With regard to toddlers and adults, the contribution of diet to total intake is high in Germany and the UK, while in the US, the high concentrations of PBDE in dust resulted in a notably smaller proportion of the intake being attributed to diet.

Comparative assessment of the inhibition of recombinant human CYP19 (aromatase) by azoles used in agriculture and as drugs for humans.

Azoles (imidazoles and triazoles) are used as antifungal agents in agriculture and in medicine, and also for antiestrogen therapy, e.g., for breast cancer treatment. Antifungal activity is based on inhibition of fungal CYP51 (lanosterol 14α‐demethylase), and estrogen biosynthesis reduction is due to azole inhibition of CYP19 (aromatase). Inhibition of aromatase by antifungal agents is usually an unwanted side effect and may cause endocrine disruption. A fluorimetric assay based on human recombinant CYP19 enzyme with dibenzylfluorescein as a substrate was used to compare the inhibitory potency of 22 azole compounds. Dose responses were established and duplicate datasets were analyzed with a nonlinear mixed‐effects model with cumulative normal distribution for the logarithm of concentration. IC50 values (50% inhibitory concentration) of 13 fungicides used in agriculture ranged more than 700‐fold, starting from 0.047 µM. The potency of seven human drugs spanned more than 7000‐fold, starting from 0.019 µM. Most potent fungicides included prochloraz, flusilazole, and imazalil, and most potent medicinal antifungals were bifonazole, miconazole, and clotrimazole. These in vitro data indicate that the top‐ranking azoles used as antifungal agents or drugs are as potent inhibitors of aromatase as are antiestrogen therapeutics used to treat breast cancer. These putative effects of azole agents and drugs on steroid biosynthesis and sex hormone balance should be considered when used in human subjects and also in wildlife exposed to azole fungicides used in agriculture.

Toxicokinetics of the mycotoxin ochratoxin A in F 344 rats after oral administration.

Ochratoxin A (OTA), a mycotoxin produced by several fungi of Aspergillus and Penicillium species, is a nephrotoxin and a renal carcinogen in rodents. This study was performed to investigate the biotransformation and toxicokinetics of this important food contaminant. Male (n=18) and female (n=18) F344 rats were administered a single dose of OTA (0.5 mg/kg b.w.) in corn oil by gavage. Animals (n=3) were sacrificed 24, 48, 72, 96, 672, and 1,344 hours after OTA administration and concentrations of OTA and OTA-metabolites in urine, feces, blood, liver, and kidney were determined by HPLC with fluorescence detection and/or by LC-MS/MS. Recovery of unchanged OTA in urine amounted to 2.1% of dose in males and 5.2% in females within 96 h. In feces, only 5.5% respectively 1.5% of dose were recovered. The major metabolite detected was OTalpha; low concentrations of OTA-glucosides were also present in urine. The maximal blood levels of OTA were observed between 24 and 48 h after administration and were appromixately 4.6 micromol/l in males and 6.0 micromol/l in females. Elimination of OTA from blood followed first-order kinetics with a half-life of approximately 230 h. In liver of both male and female rats, OTA-concentrations were less than 12 pmol/g tissue, with a maximum at 24 h after administration. In contrast, OTA accumulated in the kidneys, reaching a concentration of 480 pmol/g tissue in males 24 h after OTA-administration. Generally, tissue concentrations in males were higher than in females. OTalpha was not detected in liver and kidney tissue of rats administered OTA, and the OTalpha concentrations in blood were low (10-15 nmol/l). The high concentrations of OTA in kidneys of male rats may, in part, explain the organ- and gender-specific toxicity of OTA.

Determination of free and total bisphenol A in urine of infants

Infants may be particularly sensitive regarding hormonally active compounds such as Bisphenol A (BPA), which is widely distributed and exhibits weak oestrogenic activity. Since only free (unconjugated) BPA exhibits endocrine activity, both free and total (after hydrolysis of conjugates) BPA were determined in urine samples of infants to support valid risk assessments. Free BPA was observed above the LOQ in only 3 of 91 (3%) samples from 47 infants. As total BPA was observed in only 38 (42%) urine samples, with concentrations between <LOQ and 17.85 μg/l, the median concentration was below the LOQ of 0.45 μg/l. Based on 0.225 μg/l (half of the LOQ), a margin of exposure 1400-fold below the tolerable daily intake (TDI) of 50 μg/kg bw was calculated. The highest concentration observed (17.85 μg/l) was 18-fold below the TDI. Infants who were fed using baby bottles show approximately 2-fold higher median levels of total BPA. Importantly, the higher levels of total BPA compared to free BPA demonstrate that conversion of BPA to biologically inactive conjugates such as glucuronides or sulphates, which is well known to efficiently occur in adults, also occurs in infants.

Organophosphate flame retardants and plasticizers in the air and dust in German daycare centers and human biomonitoring in visiting children (LUPE 3)

Organophosphate (OP) flame retardants and plasticizers are chemicals that have been used in large quantities in diverse consumer and building-related products for decades. In the present study, OPs were measured in paired indoor air and dust samples from 63 daycare centers in Germany. Moreover, the urine of 312 children between 22 and 80 months old who attend these facilities was analyzed for the presence of eight OP metabolites. Tri-(2-butoxyethyl)-phosphate (TBEP), tris-(2-chloroisopropyl) phosphate (TCPP), and tri-n-butyl-phosphate (TnBP) were present in low concentrations in indoor air, with median values of 49 ng/m(3), 2.7 ng/m(3), and 2.2 ng/m(3), respectively. In dust, median values of 225 mg/kg for TBEP, 2.7 mg/kg for TCPP, 1.1mg/kg for diphenyl(2-ethylhexyl) phosphate, and 0.5mg/kg for tri-phenyl-phosphate (TPhP) were found. In the urine samples, the metabolites di-phenyl-phosphate, di-n-butyl-phosphate, and di-(2-butoxyethyl)-phosphate had median values (95th percentiles) of 0.8 μg/l (4.0 μg/l), 0.2 μg/l (0.9 μg/l), and 2.0 μg/l (10.7 μg/l), respectively. A significant correlation was found between the dust and air samples in the levels of TnBP, tris(2-chloroethyl) phosphate (TCEP), and TBEP. For TCEP and TBEP, significant correlations were also observed between the levels in dust and the respective metabolite levels in urine. For TCEP, there was also a significant correlation between the concentration in indoor air and metabolite levels in urine. Based on the 95th percentile in dust and air in our study and data from residences in a previously published study, the daily intake of the most abundant OP (TBEP) is high (i.e., 3.2 μg/kg b.w.). This level is approximately 6.4% of the reference dose (RfD) established by the NSF, U.S.A. Overall, our study shows that daycare centers are indoor environments that contribute to OP exposure.

Increased brain levels of 4-hydroxy-2-nonenal glutathione conjugates in severe Alzheimer's disease.

In the last decade an important role for the progression of neuronal cell death in Alzheimers disease (AD) has been ascribed to oxidative stress. trans-4-Hydroxy-2-nonenal, a product of lipid peroxidation, forms conjugates with a variety of nucleophilic groups such as thiols or amino moieties. Here we report for the first time the quantitation of glutathione conjugates of trans-4-hydroxy-2-nonenal (HNEGSH) in the human postmortem brain using the specific and very sensitive method of electrospray ionization triple quadrupole mass spectrometry (ESI-MS-MS). Levels of HNEGSH conjugates calculated as the sum of three chromatographically separated diastereomers were determined in hippocampus, entorhinal cortex, substantia innominata, frontal and temporal cortex, as well as cerebellum from patients with AD and controls matched for age, gender, postmortem delay and storage time. Neither age, nor postmortem delay, nor storage time did correlate with levels of HNEGSH conjugates which ranged between 1 and 500 pmol/g fresh weight in the brain areas examined. The brain specimen from patients with clinically and neuropathologically probable AD diagnosed according to criteria of the consortium to establish a registry for AD (CERAD) show increased levels of HNEGSH in the temporal and frontal cortex, as well as in the substantia innominata. Classification of disease severity according to Braak and Braak, which takes into consideration the amount of neurofibrillary tangles and neuritic plaques, revealed highest levels of HNEGSH in the substantia innominata and the hippocampus, two brain regions known to be preferentially affected in AD. These results substantiate the link between conjugates of glutathione with a product of lipid peroxidation and Alzheimers disease and justify further studies to evaluate the role of HNE metabolites as potential biomarkers for disease progression in AD.