Roman Wodarz

Azo dyes and carcinogenic aromatic amines in cell cultures

Abstract Azo dyes are widely used in the food, pharmaceutical, cosmetic, textile and leather industries. They can be reduced by azoreductases in intestinal bacteria, liver cells and skin surface microflora so that aromatic amines are released. In this study an analytical system for the determination of carcinogenic aromatic amines at the picogram to femtogram level and a cell culture assay to evaluate the toxicological effects of azo dyes and aromatic amines is presented. For the assays, the commercial azo dye Resacor Blue 2F (Color Index: Direct Blue 15), a 3,3′-dimethoxybenzidine-based dye, was used. The released carcinogenic aromatic amine, 3,3′-dimethoxybenzidine, was extracted with diethylether derivatized with pentafluoropropionic anhydride and analyzed by capillary gas chromatography/mass spectrometry. In addition, the cytotoxicity of Resacor Blue 2F on cultured kidney epithelial cells and on two hepatocyte cell lines (Hep G2 and Chang liver) was evaluated. For this purpose, the cells were exposed for 72 h to varying concentrations of Resacor Blue 2F. The results show that the azo dye inhibits the proliferation of the kidney epithelial cells much more than the proliferation of the two hepatocyte cell lines. As calculated from the dose-response curves, the EC50 (effective concentration reducing cell proliferation by 50%) for kidney epithelial cells was 40 μg/ml, whereas the EC50 for both hepatocyte cell lines was more than 250 μg/ml. Higher concentrations of 3,3′-dimethoxybenzidine were found in kidney epithelial and in Hep G2 culture supernatants; only small amounts were found in the Chang liver culture supernatant. In summary, it was demonstrated that released 3,3′-dimethoxybenzidine was found in the cell culture supernatants, but it did not accumulate within the cells. For an interpretation of the toxicological results, cell-specific transport systems and osmotic effects of the commercial azo dye which contains several inorganic and organic additives has to be considered.

Metabolism of toluene in man: gas-chromatographic determination of o-, m- and p-cresol in urine

Complete separation of phenol, o-, m- and p-cresol was achieved by capillary gas-chromatography. Urinary concentrations of cresols were determined quantitatively in samples from 10 male workers exposed to toluene. Besides pcresol, o- and m-cresol were found to be urinary compounds in the case of the exposed group in contrast to normal persons. This finding was proved by gas-chromatography/mass spectrometry. The difference between both groups is significant. It is concluded that besides hippuric acid o-, m- and p-cresol are metabolites of toluene.

Styrene metabolism in man: gas chromatographic separation of mandelic acid enantiomers in the urine of exposed persons

Abstractd- and l-mandelic acid are separated by gas chromatography as isopropyl ester or isopropyl ester-isopropyl urethane on capillary columns, coated with Chirasil-Val. For its determination in urine the isopropyl ester procedure gives better results because other components of the urine do not cause interference. Thus d- and l-mandelic acid in the urine of exposed workers could be detected and verified by means of GC/MS. Occupational styrene exposure near the MAK-value (100 ppm) results in a l/d-enantiomer-ratio in urine of nearly 1.5.

Determination of butoxyacetic acid and N-butoxyacetyl-glutamine in urine of lacquerers exposed to 2-butoxyethanol

SummaryTo determine the fraction of butoxyacetic acid (BAA) which is excreted as the amino acid conjugate N-butoxyacetylglutamine (BAA-GLN), urine samples of six lacquerers exposed to 2-butoxyethanol (BE) were collected before and after work and analysed using an HPLC-method which allows the simultaneous quantification of both BAA species. Whereas the pre-shift samples contained only little or no butoxyethanol-related material, concentrations of BAA and BAA-GLN amounted collectively to up to 7 mmol/l in the samples obtained at the end of work. The ratio BAA-GLN vs. total BAA ranged from 0.16 to 0.64 (mean value 0.48) indicating that a substantial fraction of BAA was eliminated as the amino acid conjugate. The results demonstrate that BAA-GLN is an important metabolite of BE in man. Procedures employed for the biological monitoring of exposure to BE should therefore include the quantification of BAA-GLN, otherwise exposure levels would be underestimated.

PVC-plasticizer DEHP in medical products: do thin coatings really reduce DEHP leaching into blood?

The hemocompatibility of artificial surfaces in extracorporeal blood circulation systems can be improved by coatings. According to the literature, heparin coatings should avoid the leaching of the plasticizer di(2-ethylhexyl) phthalate (DEHP) into the blood from components made from plasticized polyvinyl chloride (PVC). DEHP and its metabolites are known to impair the fertility of male rodents; effects on human fertility are assumed. Three different surface coatings with and without heparin were examined in a Chandler Loop model at 37°C using fresh human blood to evaluate their hemocompatibility and barrier property to plasticizer. The levels of toxic oxidation products of DEHP generated in the blood, particularly, were found as high as in the uncoated tubing. The coatings improved the hemocompatibility, but are not safe protection against the hazardous metabolites of DEHP. For pregnant women, neonates and children, we would recommend using the available surface-coated plasticized PVC tubing sets, but free of DEHP.

Stereometabolism of styrene in man: gas chromatographic determination of phenylethyleneglycol enantiomers and phenylethanol isomers in the urine of occupationally-exposed persons.

A gas Chromatographic procedure for the determination of phenylethyleneglycol enantiomers and phenylethanol isomers is described and applied to the investigation of urine samples from occupationally styrene-exposed workers (11 males, six females) and an unexposed control group. Phenylethyleneglycol enantiomers and 2-phenylethanol were present in the urine samples of exposed and unexposed individuals whereas 1-DL-phenylethanol was not found in control urine. The L/D enantiomer ratio of phenylethyleneglycol was found to be approximately 3 in the exposed group and 1.5 in the control group. Because of the close structural relation of these metabolites to the primarily formed epoxide, the results give further insight into the stereotoxicity of styrene in man

Biomonitoring of the di(2-ethylhexyl) phthalate metabolites mono(2-ethyl-5-hydroxyhexyl) phthalate and mono(2-ethyl-5-oxohexyl) phthalate in children and adults during the course of time and seasons

Di(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer with ubiquitous spread. Its metabolites are suspected to impair endocrine functions and fertility in man. This study is aimed to assess the DEHP exposure in children and adults, to evaluate the intraindividual variability, the influence of seasons and to identify potential routes of intake. Urine samples were collected from 6 people (age 4-58) over 7 consecutive days 4-times during a year to test for seasonal factors. 5-OH-MEHP and 5-oxo-MEHP were quantified by GC-MS. Urine concentrations of both metabolites were highly correlated with each other. Both female subjects showed remarkably low and stable 5-OH-MEHP concentrations throughout the year (median <or=64.1 and <or=78.5microg/l). Also both male adults exhibited a low burden during most measurements (median <or=50.0 and <or=52.5microg/l) except in January for the 19 year old (median: 141.4microg/l) and two single high events for the 58 year old male. In contrast, both examined boys reached weekly median concentrations as high as 171.0microg/l accompanied by a high degree of variability. Five peak events were identified, 3 of which are suspected to be caused by particular meals. During the 28 days of monitoring one adult male and two male children exceeded the tolerable metabolite concentration for the sum of 5-oxo-MEHP and 5-OH-MEHP (HBM I value) 4 times. Two of the children contributed one transgression event each, exceeding the HBM I value by a factor of 2-4. Across the 6 studied individuals we found a weak seasonal trend towards higher levels of exposure during the winter.

Bestimmung von freiem Pentachlorphenol in der Luft und im Blut durch leistungsfähige Routineverfahren

SummaryEfficient chromatographic procedures for the determination of free pentachlorophenol in air and in blood have been developed. Free pentachlorophenol from the indoor environment is preconcentrated in an impinger. Following a simple extraction, it is detected by high performance liquid chromatography without derivatisation. An alternate method is capillary gas chromatography of a suitable derivative. Unchanged pentachlorophenol in whole blood is similarly derivatised and determined quantitatively by capillary gas chromatography using an electron capture detector. The calibration curves of the described chromatographic methods were linear within the specified concentration range. Reproducibility of gas chromatographic separation remained unchanged during the whole research period. Owing to its relative simplicity, the described gas chromatographic method is advantageous for the biological monitoring of large collectives.ZusammenfassungFür die Bestimmung von freiem Pentachlorphenol in der Luft und im Blut wurden leistungsfähige chromatographische Routineverfahren ausgearbeitet. Für die Raumluftbestimmung wird Pentachlorphenol durch Einleiten von Luft in einer Vorlage angereichert und nach Aufarbeitung in freier Form durch Hochdruck-Flüssigkeits-Chromatographie quantitativ erfaßt. Alternativ kann die Analyse nach Derivatisierung durch Kapillar-Gaschromatographie erfolgen. Freies Pentachlorphenol im Vollblut wird nach Aufarbeitung und Derivatisierung durch Kapillar-Gaschromatographie in Verbindung mit dem Elektroneneinfang-Detektor quantitativ bestimmt. Bei den beschriebenen chromatographischen Methoden sind die Eichkurven über den angegebenen Konzentrationsbereich linear. Die gaschromatographische Trennung blieb über den gesamten Untersuchungszeitraum völlig reproduzierbar. Die problemlose Derivatisierung läßt die beschriebene gaschromatographische Methode in Verbindung mit einem automatischen Probengeber für das biological monitoring auch größerer Kollektive geeignet erscheinen.

Stereometabolism of ethylbenzene in man: gas chromatographic determination of urinary excreted mandelic acid enantiomers and phenylglyoxylic acid and their relation to the height of occupational exposure.

SummaryEthylbenzene is an important industrial solvent and a key substance in styrene production. Ethylbenzene metabolism leads to the formation of mandelic acid, which occurs in two enantiomeric forms, and phenyl-glyoxylic acid. To decide which enantiomer is preferably formed, 70 urine samples of exposed workers were taken at the end of shifts and — after 3-pentyl ester derivatisation — gas chromatographically analysed. The R/S ratio of mandelic acid enantiomers in urine amounts to 19:1, which means that R-mandelic acid is a major metabolite and S-mandelic acid is one of the minor urinary metabolites of ethylbenzene in man. The R/S ratio is independent of ambient air concentration of ethylbenzene within the investigated range. Compared to an ethylbenzene monoexposure the height of total mandelic acid excretion is decreased in the case of coexposure to other aromatic solvents.

Stereometabolism of styrene in man

Chiral styrene metabolites obtained during initial styrene exposure of test persons were determined in urine samples using capillary gas chromatography. A typical time-dependent urinary concentration profile of one person over a 49-h period is presented and compared with the results of a previous study of occupationally exposed workers and an unexposed control group. Maximum levels of excretion of all styrene metabolites were observed at about the end of a 9-h workshift. Forty hours after exposure, the L/D-ratio of mandelic acid had subsided to the initial value, and the L/D-ratio of phenylethylene glycol to a value equal or slightly above the initial value.