LUMOS - A Sensitive and Reliable Optode System for Measuring Dissolved Oxygen in the Nanomolar Range

Most commercially available optical oxygen sensors target the measuring range of 300 to 2 μmol L^-1. However these are not suitable for investigating the nanomolar range which is relevant for many important environmental situations. We therefore developed a miniaturized phase fluorimeter based measurement system called the LUMOS (Luminescence Measuring Oxygen Sensor). It consists of a readout device and specialized “sensing chemistry” that relies on commercially available components. The sensor material is based on palladium(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorphenyl)-porphyrin embedded in a Hyflon AD 60 polymer matrix and has a K_SV of 6.25 x 10^-3 ppmv^-1. The applicable measurement range is from 1000 nM down to a detection limit of 0.5 nM. A second sensor material based on the platinum(II) analogue of the porphyrin is spectrally compatible with the readout device and has a measurement range of 20 μM down to 10 nM. The LUMOS device is a dedicated system optimized for a high signal to noise ratio, but in principle any phase flourimeter can be adapted to act as a readout device for the highly sensitive and robust sensing chemistry. Vise versa, the LUMOS fluorimeter can be used for read out of less sensitive optical oxygen sensors based on the same or similar indicator dyes, for example for monitoring oxygen at physiological conditions. The presented sensor system exhibits lower noise, higher resolution and higher sensitivity than the electrochemical STOX sensor previously used to measure nanomolar oxygen concentrations. Oxygen contamination in common sample containers has been investigated and microbial or enzymatic oxygen consumption at nanomolar concentrations is presented.     

Evidence for toxic effects of alkylphenols from Ginkgo biloba in the hen's egg test (HET).

Extracts from the leaves of the Gingko tree (Ginkgo biloba L.) are therapeutically used for the treatment of peripheral and cerebral vascular disorders as well as multi-infarct or Alzheimer-type dementia. As constituents with potential contact allergenic and toxic properties in crude Ginkgo extracts a group of alkylphenols (e.g., ginkgolic acids, ginkgol, bilobol) has been described. Thus, for reasons of drug safety a maximal concentration (< or = 5 ppm) of ginkgolic acids is requested by the Monograph of the Commission E of the former German Federal Health Agency (Bundesgesundheitsamt, BGA). During production of the standardized Ginkgo extract EGb 761, alkylphenols are largely eliminated as water insoluble compounds (decanter sludge) from the primary acetone extract. To further assess the adverse properties of alkylphenols, different fractions derived from the decanter sludge were evaluated for their embryotoxic effects in the hen's egg test (HET). A fraction enriched for ginkgolic acids (16%) and biflavones (6.7%) was found to induce death of 50% of the chick embryos (LD50) at a dose of 1.8 mg/egg (approximately/= 33 ppm). A similar strong lethal effect (LD50: 3.5 mg/egg; 64 ppm) was oberserved for a fraction which contained 58% ginkgolic acids but less than 0.02% biflavones. In contrast, an extreme low toxic potential (LD50: 250 mg/egg or 4540 ppm) was established for a fraction containing 16% biflavones and 1% ginkgolic acids. Thus, the present investigations confirm the high toxic potential of ginkgolic acids, although it can not be excluded that biflavones or some other constituents in the different fractions may amplify the adverse effect of these substances. Since no contribution of alkylphenols to the therapeutic efficacy of Ginkgo extracts has been confirmed and their elimination during the manufacturing process does not cause technical problems, these results further support the requirement for the completest possible removal of these compounds under toxicological considerations.     

Biosensor Determination of the Microscale Distribution of Nitrate, Nitrate Assimilation, Nitrification, and Denitrification in a Diatom-Inhabited Freshwater Sediment

High-resolution NO₃⁻ profiles in freshwater sediment covered with benthic diatoms were obtained with a new microscale NO₃⁻ biosensor characterized by absence of interference from chemical species other than NO₂⁻ and N₂O. Analysis of the microprofiles obtained indicated no nitrification during darkness, high rates of nitrification and a tight coupling between nitrification and denitrification during illumination, and substantial rates of NO₃⁻ assimilation during illumination. Nitrification during darkness could be induced by purging the bulk water with O₂ gas, indicating that the stimulatory effect on nitrification by illumination was caused by algal production of O₂. NH₄⁺ addition did not stimulate nitrification during darkness when O₂ was restricted to the upper 1-mm layer, and there was thus a low nitrification potential in the permanently oxic top 1 mm of the sediment.     

Combined Micro-PET/Micro-CT Imaging of Lung Tumours in SPC-raf and SPC-myc Transgenic Mice

Introduction

SPC-raf and SPC-myc transgenic mice develop disseminated and circumscribed lung adenocarcinoma respectively, allowing for assessment of carcinogenesis and treatment strategies. The purpose of this study was to investigate the technical feasibility, the correlation of initial findings to histology and the administered radiation dose of combined micro-PET/micro-CT in these animal models.

Material and Methods

14 C57BL/6 mice (4 nontransgenic, 4 SPC-raf transgenic, 6 SPC-myc transgenic) were examined using micro-CT and ¹⁸F-Fluoro-deoxyglucose micro-PET in-vivo. Micro-PET data was corrected for random events and scatter prior to reconstruction with a 3D-FORE/2D-OSEM iterative algorithm. Rigid micro-PET/micro-CT registration was performed. Tumour-to-non-tumour ratios were calculated for different lung regions and focal lesions. Diffuse tumour growth was quantified using a semiautomated micro-CT segmentation routine reported earlier. Regional histologic tumour load was assessed using a 4-point rating scale. Gamma radiation dose was determined using thermoluminescence dosimeters.

Results

Micro-CT allowed visualisation of diffuse and circumscribed tumours in SPC-raf and SPC-myc transgenic animals along with morphology, while micro-PET provided information on metabolism, but lacked morphologic detail. Mean tumour-to-non-tumour ratio was 2.47 for circumscribed lesions. No significant correlation could be shown between histological tumour load and tumour-to-nontumour ratio for diffuse tumours in SPC-raf transgenic animals. Calculation of the expected dose based on gamma dosimetry yielded approximately 140 mGy/micro-PET examination additional to approximately 200 mGy due to micro-CT.

Conclusions

Combined micro-PET/micro-CT imaging allows for in-vivo assessment of lung tumours in SPC-raf and SPC-myc transgenic mice. The technique has potential for the evaluation of carcinogenesis and treatment strategies in circumscribed lung tumours.     

The hen's egg test for micronucleus induction (HET-MN): novel analyses with a series of well-characterized substances support the further evaluation of the test system

The hen's egg test for micronucleus induction (HET-MN) combines the use of the commonly accepted genetic endpoint "formation of micronuclei" with the well-characterized and complex model of the incubated hen's egg, which enables metabolic activation, elimination and excretion of xenobiotics -- including those that are mutagens or promutagens. This assay procedure is in line with demands for animal protection. In three previous publications we presented the scientific rationale and methodological aspects for this assay as well as results for some well-characterized mutagens and promutagens. Here we present the results of new experiments involving further genotoxic and non-genotoxic model substances. Making a comparison with published data we have to date not found any false negatives or false positives in the experiments presented here and in trials published before, thus demonstrating a promising predictivity of genotoxic effects with this assay. We could confirm relevant genotoxicity for the following substances in the HET-MN: acetylamino-fluorene (2-AAF), acrylamide (ACM), cytarabine (AraC), methotrexate (MTX), cadmium chloride (CD), dipotassium monochromate (DPC), and epirubicine (EPI). Negative results were obtained for azorubin (E122), orange G (OG) and starch (STRC). The micronucleus frequencies (MNE II) of the concurrent negative controls were in agreement with the values of the historical negative control (0.87 per thousand+/-0.87; average+/-s.d.). This value is based upon the scoring of 556,500 erythrocytes from 445 eggs. In historical positive controls the administration of 0.05mg cyclophosphamide/egg at d8 resulted in an MNE II-frequency of 12.4 per thousand+/-6.8 (average+/-s.d.) at d 10.5. This value is based upon the scoring of 249,250 erythrocytes from 223 eggs.     

Some new methodological aspects of the hen's egg test for micronucleus induction (HET-MN)

In a previous publication we introduced the hen's egg test for micronucleus induction (HET-MN) as an extremely simple, inexpensive and rapid animal free genotoxicity assay which is positioned between pure in vitro and in vivo assays, strictly in line with animal protection regulations and ethical aspects. The HET-MN combines the use of the commonly accepted genetic endpoint "formation of micronuclei" with the well characterized and complex model of the chick embryo. The high metabolic competency provided by this model enables metabolic activation, elimination and excretion of xenobiotics including mutagens and promutagens.In this paper we present some new methodological aspects, which are important for improving the experimental protocol. We used cyclophosphamide (CP) and 7,12-dimethyl-benz[a]anthracene (DMBA) as model substances. Dose-response-relationship for both chemicals and cytotoxic effects for CP are described. In addition to the standard proliferation marker PCE/NCE-ratio we found an increased frequency of primitive erythrocytes (E I) and the appearance of proerythroblasts and erythroblasts as further alerting signals for cytotoxic or erythrosuppressive effects. From the total cell population we could further qualify the group of target cells. We found that all definite erythrocytes (E II), observed at day 11 (d11), are relevant target cells, independent from their stage of maturity (polychromatic as well as normochromatic definite erythrocytes). E I cells do not belong to the group of target cells, however. An additional important methodological aspect is the optimal time frame. We found the time period from d8 of incubation (administration of the test substance) up to d11 (time point of blood sampling) as most favorable. In this way an exposure period of up to 72h is covered. Further results indicate that the air cell route provides a higher response to the test substances than the albumen route. The consideration of the described methodological aspects will contribute to the improvement of the experimental protocol of the HET-MN.     

Confirmation of 'aerobic denitrification' in batch cultures, using gas chromatography and 15N mass spectrometry

Abstract: Because of a revival in the controversy surrounding 'aerobic denitrification', especially in relation to Thiosphaera pantotropha , activity in aerobic batch cultures was evaluated using gas chromatography and mass spectrometry after the addition of ¹⁵N-labelled NH₄⁺ and NO₂⁻. Aerobic denitrifying activity in T. pantotropha was present, but only at about 10% of the originally-reported levels. The activity of ' Pseudomonas denitrificans ' was similar to previously-reported values. Alcaligenes faecalis showed significant aerobic denitrifying activity, producing almost equivalent amounts of N₂ and N₂O. An unidentified pseudomonad, isolate G4, presumably requires anoxia for enzyme activity as it did not denitrify aerobically, even though it has a constitutive denitrifying pathway.     

The hen's egg test for micronucleus induction (HET-MN): Novel analyses with a series of well-characterized substances support the further evaluation of the test system

The hen's egg test for micronucleus induction (HET-MN) combines the use of the commonly accepted genetic endpoint “formation of micronuclei” with the well-characterized and complex model of the incubated hen's egg, which enables metabolic activation, elimination and excretion of xenobiotics—including those that are mutagens or promutagens. This assay procedure is in line with demands for animal protection. In three previous publications we presented the scientific rationale and methodological aspects for this assay as well as results for some well-characterized mutagens and promutagens. Here we present the results of new experiments involving further genotoxic and non-genotoxic model substances. Making a comparison with published data we have to date not found any false negatives or false positives in the experiments presented here and in trials published before, thus demonstrating a promising predictivity of genotoxic effects with this assay.We could confirm relevant genotoxicity for the following substances in the HET-MN: acetylamino-fluorene (2-AAF), acrylamide (ACM), cytarabine (AraC), methotrexate (MTX), cadmium chloride (CD), dipotassium monochromate (DPC), and epirubicine (EPI). Negative results were obtained for azorubin (E122), orange G (OG) and starch (STRC).The micronucleus frequencies (MNE II) of the concurrent negative controls were in agreement with the values of the historical negative control (0.87‰ ± 0.87; average ± s.d.). This value is based upon the scoring of 556,500 erythrocytes from 445 eggs. In historical positive controls the administration of 0.05 mg cyclophosphamide/egg at d8 resulted in an MNE II-frequency of 12.4‰ ± 6.8 (average ± s.d.) at d 10.5. This value is based upon the scoring of 249,250 erythrocytes from 223 eggs.