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Andreas Hartmann

Publications and source records attributed to Andreas Hartmann.

35 records · Page 2Linked to original sources
PubMed2005

The comet assay: a sensitive genotoxicity test for the detection of DNA damage.

The comet assay or single-cell gel (SCG) test is a microgel electrophoresis technique that measures DNA damage at the level of single cells. A small number of cells suspended in a thin agarose gel on a microscope slide is lysed, electrophoresed, and stained with a fluorescent DNA binding dye. Cells with increased DNA damage display increased migration of chromosomal DNA from the nucleus toward the anode, which resembles the shape of a comet. In its alkaline version, which is mainly used, DNA single-strand breaks, DNA double-strand breaks, alkali-labile sites, and single-strand breaks associated with incomplete excision repair sites cause increased DNA migration. On the other hand, crosslinks (DNA-DNA or DNA-protein) can lead to decreased DNA migration. Variations of the comet assay have been established for the detection of specific DNA base modifications. Here we describe the basic methodology of the alkaline comet assay, establishing a sensitive protocol for obtaining reproducible and reliable data. Applications of the comet assay for detecting DNA damage in individual cells are briefly reviewed.

Animals↗
PubMed2004

[Current aspects of electrical cardioversion in patients with persistent atrial fibrillation].

BACKGROUND: Results of recent studies suggest new aspects in the concept of the management of patients with atrial fibrillation. Current trials comparing rhythm control by cardioversion followed by antiarrhythmic drug therapy with heart rate control only showed that rhythm control was not superior to rate control. SPECIAL ASPECTS OF ELECTRICAL CARDIOVERSION: However, some subgroups of patients with frequent or severe symptoms and younger patients with structurally normal hearts may benefit from cardioversion. In patients with persistent atrial fibrillation, sinus rhythm can be restored in approximately 90% by direct current transthoracic cardioversion. Transthoracic cardioversion is effective and safe, if anticoagulation is used. Attempts have been made to improve the success rate by new techniques. However, definition of subgroups and stabilization of sinus rhythm are ongoing issues of debate.

Anti-Arrhythmia Agents↗
PubMed2004

Gene expression profiles derived from single cells in human postmortem brain.

The study of postmortem human brain tissue remains the basis for the understanding of many CNS disorders and to verify data obtained in experimental studies. So far, however, gene expression profiling in cellular sub-populations derived from human postmortem brain was hampered by several technical drawbacks. Here, we describe a method that allows the generation of mRNA expression profiles from single neurons. Dopaminergic neurons from different midbrain areas including substantia nigra, central gray substance and ventral tegmental area were identified and isolated by immuno-laser capture microscopy (LCM). Expression profiles were generated from microdissected cells using a modified RNA fingerprinting protocol. Using this approach, we were able to generate specific RNA fingerprints at a high resolution from phenotype-specific single neurons. Polymorphic fragments were isolated from gels and differential gene expression was confirmed by real-time PCR using gene-specific primer pairs and hybridization probes. The method described here is easy to use and reliable for profiling gene expression at the single cell level in human postmortem brain. It could therefore be valuable to open new insights into the molecular pathogenesis of CNS disorders.

Aged↗
PubMed2004

Use of the alkaline in vivo Comet assay for mechanistic genotoxicity investigations.

The alkaline Comet assay was used to investigate the in vivo genotoxicity of 17 compounds. Altogether 21 studies were conducted with these compounds. The investigations were triggered for various reasons. The main reason for performing the studies was to evaluate the in vivo relevance of in vitro genotoxicity findings with 10 compounds. Eight of these compounds showed no effects in the in vivo Comet assay while two compounds induced altered DNA migration patterns in specific organs. The remaining seven compounds were tested to follow up on neoplastic/preneoplastic or chronic toxicity changes as detected in specific target organs identified in rodent studies, to investigate the possibility of site-of-contact genotoxicity and to test the liver as a target organ for a suspected reactive metabolite. For the studies, various organs of rodents were analyzed, depending on the suspected properties of the compounds, including liver, jejunum, leukocytes, stomach mucosa, duodenum, lung and kidney. All tissues were amenable to investigation by gel electrophoresis after simple disaggregation of organs by means of mincing or, in the case of epithelial cells from the gastrointestinal tract, scraping off cells from the epithelium. In conclusion, the Comet assay was found to be a reliable and robust test to investigate in vivo genotoxicity in a variety of rodent organs. Therefore, it is concluded that in vivo Comet assay data are useful for elucidating positive in vitro genotoxicity findings and to evaluate genotoxicity in target organs of toxicity.

Animals↗
PubMed2003

Comparative study with the alkaline Comet assay and the chromosome aberration test.

The alkaline Comet assay is becoming a useful tool for early genotoxicity testing of new pharmaceutical drug candidates. The aim of this study was to elucidate the predictive value of Comet assay results for the outcome of the chromosome aberration (CA) test. For this purpose, a validation exercise with 13 drug candidates was carried out utilizing V79 Chinese hamster cells and human lymphocytes. The study demonstrates that results of the Comet assay and the chromosome aberration test show a high degree of agreement, irrespective of the cell type used. In the Comet assay, seven compounds were positive and six were negative, while in the CA test, six were positive and seven were negative. The only discrepancy was found with one compound that was positive in the Comet assay with V79 cells, negative in the Comet assay with human lymphocytes and clearly negative in the CA test with human lymphocytes. For the selection of concentrations for testing in the Comet assay, cytotoxicity by means of cell count after incubation or viability by means of Trypan-blue dye exclusion (TBDE) were used. The results show that either parameter led to analysis of a concentration range in the Comet assay similar to that chosen in the CA test, in which cell count (when using V79 cells) or mitotic index (in case of lymphocytes) were used. However, since cell count after incubation of cells is much more labour-intensive, viability was preferred as the parameter to assess cytotoxicity and for selecting concentrations for analysis in the Comet assay. The data presented in this study may contribute the regulatory acceptance of the Comet assay, e.g. for mechanistic studies.

Animals↗
PubMed2003

Relationship between selected overdrive parameters and the therapeutic outcome and tolerance of atrial overdrive pacing.

There is a paucity of information on the influence of selected overdrive parameters on the clinical efficacy and tolerance of atrial overdrive algorithms to suppress atrial tachyarrhythmias. Data from a completed clinical trial investigating a new DDD+ overdrive algorithm implemented in a permanent pacemaker were analyzed. One-hundred patients with standard pacing indications and atrial tachyarrhythmias were enrolled and followed for 6 months in DDD and 6 months in DDD+ mode in a randomized, crossover fashion. The overdrive step size was programmed at the discretion of the investigators between 4 and 12 beats/min, overdrive plateau length between 10 and 32 beats, and maximum overdrive rate between 100 and 160 beats/min. The effects of DDD+ versus DDD mode on burden and incidence of atrial tachyarrhythmias stored in the mode switch memory were examined as a function of the programmed overdrive parameters. An overdrive step size between 7 and 12 beats/min, and higher a maximum overdrive rate between 121 and 160 beats/min were slightly more effective than lower programmed values, though > 500 randomized, crossover observations would have been necessary to verify statistical significance. Overdrive pacing related symptoms mandated early manual deactivation of overdrive pacing in 4.7% of 85 evaluated patients. Overdrive was disabled without testing tolerability of less aggressive overdrive values. There was no link between patient symptoms and programmed overdrive step size or overdrive plateau length values.

Aged↗
PubMed2003

A toxicity identification evaluation approach to studying estrogenic substances in hog manure and agricultural runoff.

Spreading liquid manure on agricultural fields is a routine way of disposing of animal manure and optimizing the use of nutrients for crops. Limited studies suggest that these wastes may contain a variety of endocrine-disrupting compounds (EDCs) that may be released into aquatic environments through runoff. The purpose of this study was to apply a toxicity identification and evaluation approach to isolate and identify estrogenic compounds in hog manure. A recombinant yeast estrogen screen bioassay was used to detect estrogenicity of high-performance liquid chromatography--separated hog manure fractions. Further analytical analyses of the fractions and comparison to authentic standards resulted in the identification of the endogenous estrogens 17 beta-estradiol (E2) and estrone, and the phytoestrogen metabolite, equol. High levels of equol (6.9-16.6 ppm) were found to be present in manure that was stored for several months. The endocrine-disrupting potential of equol was characterized further by using fish hormone estrogen receptor (ER), sex hormone binding protein (SSBP), and goldfish androgen receptor (AR) radioligand binding assays. Equol was found to be approximately 1,000- and 200-fold less potent that E2 in competing for binding sites of the SSBP and ER, respectively. Equol's potency was 2,200-fold less than testosterone for the AR. Additional studies confirmed the presence of compounds with estrogenic activity in tile drain water after application of hog manure to an agriculture field. In this case, the contribution of equol to the total estrogenicity of the tile drain water was minimal relative to that of natural estrogens. Overall, this study indicates that further work is warranted to assess the impact that EDCs that originate from agricultural runoff may have on the ecology or physiology of exposed biota.

Agriculture↗
PubMed2003

A strategy to reduce the numbers of fish used in acute ecotoxicity testing of pharmaceuticals.

The pharmaceutical industry gives high priority to animal welfare in the process of drug discovery and safety assessment. In the context of environmental assessments of active pharmaceutical ingredients (APIs), existing U.S. Food and Drug Administration and draft European regulations may require testing of APIs for acute ecotoxicity to algae, daphnids, and fish (base-set ecotoxicity data used to derive the predicted no-effect concentration [PNECwater] from the most sensitive of three species). Subject to regulatory approval, it is proposed that testing can be moved from fish median lethal concentration (LC50) testing (typically using > or = 42 fish/API) to acute threshold tests using fewer fish (typically 10 fish/API). To support this strategy, we have collated base-set ecotoxicity data from regulatory studies of 91 APIs (names coded for commercial reasons). For 73 of the 91 APIs, the algal median effect concentration (EC50) and daphnid EC50 values were lower than or equal to the fish LC50 data. Thus, for approximately 80% of these APIs, algal and daphnid acute EC50 data could have been used in the absence of fish LC50 data to derive PNECwater values. For the other 18 APIs, use of an acute threshold test with a step-down factor of 3.2 is predicted to give comparable PNECwater outcomes. Based on this preliminary scenario of 91 APIs, this approach is predicted to reduce the total number of fish used from 3,822 to 1,025 (approximately 73%). The present study, although preliminary, suggests that the current regulatory requirement for fish LC50 data regarding APIs should be succeeded by fish acute threshold (step-down) test data, thereby achieving significant animal welfare benefits with no loss of data for PNECwater estimates.

Animal Testing Alternatives↗
PubMed2002

Bystander effects in radiation-induced genomic instability.

Exposure of GM10115 hamster-human hybrid cells to X-rays can result in the induction of chromosomal instability in the progeny of surviving cells. This instability manifests as the dynamic production of novel sub-populations of cells with unique cytogenetic rearrangements involving the "marker" human chromosome. We have used the comet assay to investigate whether there was an elevated level of endogenous DNA breaks in chromosomally unstable clones that could provide a source for the chromosomal rearrangements and thus account for the persistent instability observed. Our results indicate no significant difference in comet tail measurement between non-irradiated and radiation-induced chromosomally unstable clones. Using two-color fluorescence in situ hybridization we also investigated whether recombinational events involving the interstitial telomere repeat-like sequences in GM10115 cells were involved at frequencies higher than random processes would otherwise predict. Nine of 11 clones demonstrated a significantly higher than expected involvement of these interstitial telomere repeat-like sequences at the recombination junction between the human and hamster chromosomes. Since elevated levels of endogenous breaks were not detected in unstable clones we propose that epigenetic or bystander effects (BSEs) lead to the activation of recombinational pathways that perpetuate the unstable phenotype. Specifically, we expand upon the hypothesis that radiation induces conditions and/or factors that stimulate the production of reactive oxygen species (ROS). These reactive intermediates then contribute to a chronic pro-oxidant environment that cycles over multiple generations, promoting chromosomal recombination and other phenotypes associated with genomic instability.

Animals↗
PubMed2002

Genotoxicity assessment of the antiepileptic drug AMP397, an Ames-positive aromatic nitro compound.

AMP397 is a novel antiepileptic agent and the first competitive AMPA antagonist with high receptor affinity, good in vivo potency, and oral activity. AMP397 has a structural alert (aromatic nitro group) and was mutagenic in Salmonella typhimurium strains TA97a, TA98 and TA100 without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR. The amino derivative of AMP397 was negative in wild-type strains TA98 and TA100. AMP397 was negative in a mouse lymphoma tk assay, which included a 24h treatment without S9. A weak micronucleus induction in vitro was found at the highest concentrations tested in V79 cells with S9. AMP397 was negative in the following in vivo studies, which included the maximum tolerated doses of 320mg/kg in mice and 2000mg/kg in rats: MutaMouse assay in colon and liver (5x320mg/kg) at three sampling times (3, 7 and 31 days after the last administration); DNA binding study in the liver of mice and rats after a single treatment with [14C]-AMP397; comet assay (1x2000mg/kg) in jejunum and liver of rats, sampling times 3 and 24h after administration; micronucleus test (2x320mg/kg) in the bone marrow of mice, sampling 24h after the second administration. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. In particular, no genotoxic metabolite is formed in mammalian cells, and, if formed by intestinal bacteria, is unable to exert any genotoxic activity in the adjacent intestinal tissue. These data were considered to provide sufficient safety to initiate clinical development of the compound.

Animals↗
PubMed2002

Role of TNF-alpha receptors in mice intoxicated with the parkinsonian toxin MPTP.

The loss of dopaminergic neurons in Parkinson's disease is associated with a glial reaction and the overproduction of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha). TNF-alpha acts via two different receptors, TNFR1 and TNFR2, and is believed to have both a neuroprotective and a deleterious role for neurons. In order to analyze the putative role of TNF-alpha in parkinsonism, we compared the effect of the parkinsonian drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice lacking TNFR1, TNFR2, or both receptors and in wild-type littermates. We show that MPTP does not affect spontaneous activity or anxiety in any of the groups and that it reduces motor activity on a rotarod in double knock out mice but not in mice lacking only one receptor. Postmortem analysis revealed no differences in the number of nigral dopaminergic neurons whatever the group. In contrast, striatal dopamine level was slightly decreased in double knock-out mice and more reduced by MPTP in this group than in the other groups of mice. In addition, dopamine turnover was significantly more increased in double knock out mice after MPTP injection. These data suggest that TNF-alpha does not participate in the death of dopaminergic neurons in parkinsonism but that it slightly alters dopamine metabolism or the survival of dopaminergic terminals by a mechanism involving both receptors.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2002

Increased expression and redistribution of the antiapoptotic molecule Bcl-xL in Parkinson's disease.

In the present study, we tried to clarify the potentially protective role of Bcl-x(L), an anti-apoptotic member of the Bcl-2 family of proteins, in Parkinson's disease (PD). Using in situ hybridization on human postmortem mesencephalon sections, we show that in PD patients Bcl-x(L) mRNA expression per dopaminergic neuron was almost double that of controls. We also show that, ultrastructurally, this effect may be mediated by a redistribution of Bcl-x(L) from the cytosol to the outer mitochondrial membrane.

Aged↗
PubMed2002

Rate-adaptive pacing using intracardiac impedance shows no evidence for positive feedback during dobutamine stress test.

BACKGROUND: The Inos2 DDDR pacemaker senses unipolar intracardiac impedance signals for adapting heart rate to meet the haemodynamic needs of physical activity. Theoretically, such pacing devices could be limited by positive feedback since increase in beating frequency per se enhances contractility. We have addressed this problem in patients with chronotropic incompetence who were subjected to a pharmacological stress test. METHODS AND RESULTS: Twelve patients with chronically implanted Inos2 DDDR pacemakers were studied using the standard protocol of stress echocardiography. Most of the patients reached the programmable maximum closed-loop rate during the incremental dobutamine challenge. The time courses for increase in as well as for recovery of heart rate were not different from control patients receiving routine diagnostic stress echocardiography. CONCLUSIONS: In patients implanted with the Inos2 DDDR pacemaker acute stimulation with intravenous dobutamine leads to a robust increase in paced heart rate without any evidence of positive feedback.

Cardiac Pacing, Artificial↗
PubMed2002

High throughput Comet assay using 96-well plates.

The single-cell gel electrophoresis or Comet assay is becoming established as an industrial genotoxicity screening test. The aim of this study was to increase the throughput of compounds tested and to minimize the amount of test compound needed for an assay. We modified practical aspects of our standard protocol and designed an experimental procedure suitable for use with 96-well plates. By using a suspension culture rather than attached cells, the modified protocol enabled parallel testing of four compounds on a single microplate (10 duplicate concentrations per compound). A significant reduction in work time was achieved by replacing the previously used Trypan blue dye exclusion (TBDE) test by an automated measurement of ATP levels as the concurrent viability test. The rapid and easy to perform ATP test was carried out towards the end of the 3 h treatment. In this way we were able to select for further analysis and slide preparation only those concentrations which induced the desired range of cytotoxicity. The suitability of the modified test conditions and reproducibility of test results was demonstrated by results obtained with standard mutagens and eight drug candidates tested at various concentrations. In each case the results obtained with the standard and the modified protocols were comparable. By introducing the changes to our standard protocol, combined with automated image analysis, we were able to more than double our previous throughput.

Adenosine Triphosphate↗
PubMedDate not supplied

Cerebrovascular reactivity in major depression: a pilot study.

OBJECTIVE: There are a growing number of reports that depression may increase the risk of stroke. Little is known, however, about the pathophysiologic mechanisms underlying this association. Cerebrovascular reactivity (CVR) reflects the compensatory dilatory capacity of cerebral arterioles to a dilatory stimulus and is an important mechanism to provide constant cerebral blood flow. We hypothesized that CVR is reduced in patients with major depression, thus contributing to the association between depression and stroke. METHODS: We assessed CVR in 33 patients with unipolar depression and 26 healthy controls by calculating the increase in cerebral blood flow velocity after stimulation with acetazolamide. Blood flow velocities were measured by transcranial Doppler ultrasound. RESULTS: Cerebrovascular reactivity was significantly reduced in depressed patients. Smoking was also associated with a significant reduction in CVR, whereas age and gender had no significant influence. CONCLUSIONS: Cerebrovascular reactivity appears to be impaired in major depression. Further studies should clarify the mechanisms leading to this reduced CVR.

Acetazolamide↗