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1.  Exploring LA-ICP-MS as a quantitative imaging technique to study nanoparticle uptake in Daphnia magna and zebrafish (Danio rerio) embryos 
Analytical and Bioanalytical Chemistry  2015;407(18):5477-5485.
The extent and the mechanisms by which engineered nanoparticles (ENPs) are incorporated into biological tissues are a matter of intensive research. Therefore, laser ablation coupled to inductively coupled plasma mass spectrometry (LA-ICP-MS) is presented for the detection and visualization of engineered nanoparticles (Al2O3, Ag, and Au) in ecotoxicological test organisms (Danio rerio and Daphnia magna). While ENPs are not taken up by the zebrafish embryo but attach to its chorion, incorporation into the gut of D. magna is clearly visible by a 50-μm spot ablation of 40-μm-thick organism sections. During laser ablation of the soft organic matrix, the hard ENPs are mobilized without a significant change in their size, leading to decreasing sensitivity with increasing size of ENPs. To compensate for these effects, a matrix-matched calibration with ENPs of the same size embedded in agarose gels is proposed. Based on such a calibration, the mass of ENPs within one organism section was calculated and used to estimate the total mass of ENPs per organism. Compared to the amount determined after acid digestion of the test organisms, recoveries of 20–100 % (zebrafish embryo (ZFE)) and of 4–230 % (D. magna) were obtained with LODs in the low ppm range. It is likely that these differences are primarily due to an inhomogeneous particle distribution in the organisms and to shifts in the particle size distribution from the initial ENPs to those present in the organism. It appears that quantitative imaging of ENPs with LA-ICP-MS requires knowledge of the particle sizes in the biological tissue under study.
Electronic supplementary material
The online version of this article (doi:10.1007/s00216-015-8720-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s00216-015-8720-4
PMCID: PMC4477941  PMID: 25943260
Visualization; Toxicity; Accumulation; Microscopy; Tissue; Soft biological matrices
2.  Quantification of Al2O3 nanoparticles in human cell lines applying inductively coupled plasma mass spectrometry (neb-ICP-MS, LA-ICP-MS) and flow cytometry-based methods 
In order to quantify and compare the uptake of aluminum oxide nanoparticles of three different sizes into two human cell lines (skin keratinocytes (HaCaT) and lung epithelial cells (A549)), three analytical methods were applied: digestion followed by nebulization inductively coupled plasma mass spectrometry (neb-ICP-MS), direct laser ablation ICP-MS (LA-ICP-MS), and flow cytometry. Light and electron microscopy revealed an accumulation and agglomeration of all particle types within the cell cytoplasm, whereas no particles were detected in the cell nuclei. The internalized Al2O3 particles exerted no toxicity in the two cell lines after 24 h of exposure. The smallest particles with a primary particle size (xBET) of 14 nm (Alu1) showed the lowest sedimentation velocity within the cell culture media, but were calculated to have settled completely after 20 h. Alu2 (xBET = 111 nm) and Alu3 (xBET = 750 nm) were calculated to reach the cell surface after 7 h and 3 min, respectively. The internal concentrations determined with the different methods lay in a comparable range of 2–8 µg Al2O3/cm2 cell layer, indicating the suitability of all methods to quantify the nanoparticle uptake. Nevertheless, particle size limitations of analytical methods using optical devices were demonstrated for LA-ICP-MS and flow cytometry. Furthermore, the consideration and comparison of particle properties as parameters for particle internalization revealed the particle size and the exposure concentration as determining factors for particle uptake.
Electronic supplementary material
The online version of this article (doi:10.1007/s11051-014-2592-y) contains supplementary material, which is available to authorized users.
doi:10.1007/s11051-014-2592-y
PMCID: PMC4176630  PMID: 25285033
Inductively coupled plasma mass spectrometry (ICP-MS); Flow cytometry; Size dependency; Cellular internalization; Aluminum oxide
3.  Metabolism of 2-Mercaptobenzothiazole by Rhodococcus rhodochrous 
Applied and Environmental Microbiology  2004;70(10):6315-6319.
2-Mercaptobenzothiazole, which is mainly used in the rubber industry as a vulcanization accelerator, is very toxic and is considered to be recalcitrant. We show here for the first time that it can be biotransformed and partially mineralized by a pure-culture bacterial strain of Rhodococcus rhodochrous. Three metabolites, among four detected, were identified.
doi:10.1128/AEM.70.10.6315-6319.2004
PMCID: PMC522129  PMID: 15466583
4.  Benzothiazole Degradation by Rhodococcus pyridinovorans Strain PA: Evidence of a Catechol 1,2-Dioxygenase Activity 
Applied and Environmental Microbiology  2002;68(12):6114-6120.
The pathway for biodegradation of benzothiazole (BT) and 2-hydroxybenzothiazole (OBT) by Rhodococcus pyridinovorans strain PA was studied in detail. The kinetics of biodegradation were monitored by in situ 1H nuclear magnetic resonance (NMR) in parallel with reversed-phase high-performance liquid chromatography (HPLC). Successive oxidations from BT to OBT and then from OBT to dihydroxybenzothiazole were observed. Further insight was obtained by using a mutant strain with impaired ability to grow on BT and OBT. The precise structure of another intermediate was determined by in situ two-dimensional 1H-13C NMR and HPLC-electrospray ionization mass spectrometry; this intermediate was found to be a ring-opening product (a diacid structure). Detection of this metabolite, together with the results obtained by 1H and 19F NMR when cells were incubated with 3-fluorocatechol, demonstrated that a catechol 1,2-dioxygenase is involved in a pathway for biodegradation of BTs in this Rhodococcus strain. Our results show that catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities may both be involved in the biodegradation of BTs depending on the culture conditions.
doi:10.1128/AEM.68.12.6114-6120.2002
PMCID: PMC134439  PMID: 12450835
5.  Identification of Quinoide Redox Mediators That Are Formed during the Degradation of Naphthalene-2-Sulfonate by Sphingomonas xenophaga BN6 
During aerobic degradation of naphthalene-2-sulfonate (2NS), Sphingomonas xenophaga strain BN6 produces redox mediators which significantly increase the ability of the strain to reduce azo dyes under anaerobic conditions. It was previously suggested that 1,2-dihydroxynaphthalene (1,2-DHN), which is an intermediate in the degradative pathway of 2NS, is the precursor of these redox mediators. In order to analyze the importance of the formation of 1,2-DHN, the dihydroxynaphthalene dioxygenase gene (nsaC) was disrupted by gene replacement. The resulting strain, strain AKE1, did not degrade 2NS to salicylate. After aerobic preincubation with 2NS, strain AKE1 exhibited much higher reduction capacities for azo dyes under anaerobic conditions than the wild-type strain exhibited. Several compounds were present in the culture supernatants which enhanced the ability of S. xenophaga BN6 to reduce azo dyes under anaerobic conditions. Two major redox mediators were purified from the culture supernatants, and they were identified by high-performance liquid chromatography-mass spectrometry and comparison with chemically synthesized standards as 4-amino-1,2-naphthoquinone and 4-ethanolamino-1,2-naphthoquinone.
doi:10.1128/AEM.68.9.4341-4349.2002
PMCID: PMC124094  PMID: 12200285
6.  Direct Ring Fission of Salicylate by a Salicylate 1,2-Dioxygenase Activity from Pseudaminobacter salicylatoxidans 
Journal of Bacteriology  2001;183(23):6936-6942.
In cell extracts of Pseudaminobacter salicylatoxidans strain BN12, an enzymatic activity was detected which converted salicylate in an oxygen-dependent but NAD(P)H-independent reaction to a product with an absorbance maximum at 283 nm. This metabolite was isolated, purified, and identified by mass spectrometry and 1H and 13C nuclear magnetic resonance spectroscopy as 2-oxohepta-3,5-dienedioic acid. This metabolite could be formed only by direct ring fission of salicylate by a 1,2-dioxygenase reaction. Cell extracts from P. salicylatoxidans also oxidized 5-aminosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-methylsalicylate, 3- and 5-hydroxysalicylate (gentisate), and 1-hydroxy-2-naphthoate. The dioxygenase was purified and shown to consist of four identical subunits with a molecular weight of about 45,000. The purified enzyme showed higher catalytic constants with gentisate or 1-hydroxy-2-naphthoate than with salicylate. It was therefore concluded that P. salicylatoxidans synthesized a gentisate 1,2-dioxygenase with an extraordinary substrate range, which also allowed the oxidation of salicylate.
doi:10.1128/JB.183.23.6936-6942.2001
PMCID: PMC95535  PMID: 11698383

Results 1-6 (6)