COMET ASSAY embedding in agarose of blood samples and dropping on comet slides
EPrints System ErrorConceived and designed the experiments: Terahertz testosterone injection video downloader fields are non-ionizing electromagnetic fields in the frequency methyl methanesulfonate comet assay from 0. Potential applications of these electromagnetic fields include the whole body scanners, which currently apply millimeter waves just below the terahertz range, but future scanners will use higher frequencies in the terahertz range. These and other applications will bring along methanesulfonatd exposure to these fields. Up to now, only a limited number mrthanesulfonate investigations on biological effects of terahertz electromagnetic fields have been performed.
Comparative evaluation of the in vitro micronucleus test and the in vitro chromosome aberration test: Industrial experience Genotoxic effects of known and suspected aneugens: A combination of mutagenicity and recombinogenicity may confer aflatoxin B1 a potent liver carcinogen Evaluation of the comet assay as a routine in vitro genotoxicity test for surface waters Evaluation of the in vitro micronucleus test mnt as an alternative to the in vitro chromosomal aberration assay ca: Servier, F Courbevoie France.
This contribution analyses a database assembled in four industrial laboratories using the in vitro micronucleus assay in routine screening for detection of chromosomal aberrations. A comparison of data from the in vitro micronucleus assay with data of standard chromosome aberration assay strongly supports that the in vitro micronucleus test is a suitable alternative for the in vitro chromosome aberration test.
The in vitro micronucleus test has several advantages: The available data indicate, that the in vitro micronucleus test is somewhat more sensitive than the CA lowest effective doses tested lower for positive compounds.
The very high concordance is remarkable in particular taking into account that the compounds evaluated except for the gyrase and topoisomerase inhibitors are only weak inducers in general. The in vitro micronucleus test has the potential of not only detecting clastogens but in addition aneuploidy inducing chemicals. Genotoxic effects of known and suspected aneugens: The well known aneugen colchicine and the suspected aneugens chloral hydrate, hydroquinone and thimerosal are known to induce micronuclei in vitro , but data on the induction of chromosomal aberrations, polyploidy and gene mutation are inconclusive or not available.
In order to correlate the micronucleus data with other genotoxic effects, these compounds were tested in three different mammalian in vitro systems. For the micronucleus and the chromosome aberration test clone K-5 from cell line CCL 61 Chinese hamster ovary cells established in our laboratory was used. The cells were treated with the test compounds for 24 hours in the micronucleus assay and for 18, 24, 42 or 48 hours in the chromosome aberration assay.
Colchicine induced micronuclei at concentrations around 0. The compound was clastogenic at high concentrations, induced polyploidy and was negative in the mouse lymphoma test. The compound induced also polyploidy, but was negative in the gene mutation assay. Hydroquinone was slightly positive in the micronucleus and the chromosome aberration assay and it revealed distinct effects in the mouse lymphoma test. Thimerosal showed clear-cut effects in both, the micronucleus and the chromosome aberration assay, and it induced polyploidy.
It was, however, negative in the mouse lymphoma test. Micronucleus induction does not parallel the formation of structural chromosomal aberrations with colchicine. However, with chloral hydrate, hydroquinone and thimerosal both effects occurred at comparable concentrations.
Different DNA damaging species produced by radical reactions of aliphatic aldehydes with cu ll. The transition metal copper is able to induce redox reactions via one electron transfers. Thereby a well-known mechanism is the copper ll catalyzed oxidation of hydroquinones followed by a one electron transfer from the reduced copper l ion to 0 2. We found that aldehydes are oxidable by Cu ll too, resulting in excessive DNA single and double strand breakage 2.
The reaction of the isomer n-butyraldehyde with Cu ll is not to explain in this way. In addition this was confirmed by the ineffectivity of catalase to prevent DNA-strand breakage. The formation of excited species others than 1 0 2 points to fundamentally different reaction mechanism in Cu ll driven oxidations as known so far.
DNA damage resulting from the oxidation of hydroquinone by copper: Carcinogenesis, 14 7 , DNA single and double strand breaks induced by aliphatic and aromatic aldehydes in combination with copper ll. Transgenic mice as a tool to analyse the role of DNA primary lesions and 0 6 -methylguanine-DNA methyltransferase in multistage skin carcinogenesis. In order to study in vivo the contribution of 0 6 -methylguanine O 6 meG adducts to chemical skin carcinogenesis and to define the role of the DNA repair protein 0 6 -methylguanine-DNA methyltransferase MGMT in protection against tumor formation during multistage carcinogenesis, we generated transgenic mice that overexpress the human MGMT cDNA in epidermal cells by virtue of bovine cytokeratin III and IV promoter elements.
To evaluate both aspects in the process of alkylation-induced tumor initiation, transgenic and nontransgenic mice were topically treated with a single dose of 20 and 50 m mol MNU, respectively, followed by repeated application of the tumor promoter TPA. A significant and dose-dependent skin-tumor response was observed in nontransgenic mice, whereas transgenic mice showed an approx.
When DMBA, which induces mutations in mouse skin by other lesions than O 6 alkG was used for tumor initiation, no difference in tumor response of both groups of mice was observed. These data provide clear evidence that MGMT protects cells from alkylation-induced tumor initiation without affecting tumor promotion. A base substitutions in codon 12 of the Ha-ras gene were shown to be the predominating mutation responsible for MNU-induced papilloma formation.
Sequencing of Ha-ras mutations in ACNU-induced papillomas revealed a higher percentage of genetic alterations outside codon 12 as compared to MNU-induced skin tumors.
Moreover, experiments are currently being performed to gain more insights into the relevance of O 6 meG base damage and the protective function of MGMT in malignant progression of papillomas to squamous cell carcinomas. DNA repair, 0 6 -methylguanine-DNA methyltransferase, multistage carcinogenesis, skin, transgenic mice, alkylating agents. Microinjection of recombinant genes into the mouse zygote usually results in the gain of a novel function in transgenic offspring.
The promoter of the recombinant transgene determines its transcription pattern and may lead to the ectopic or over expression of a normal gene or to the de novo expression of a mutant gene in specific tissues thus recapitulating human pathophysiological processes in mouse models.
The regulated expression of transgenes by tetracycline-controllable transactivation may offer additional clues to the physiological or pathophysiological function of normal or mutant genes in vivo. The target mutagenesis of endogenous genes by homologous recombination in mouse embryonic stem cells allows the inactivation of specific genes in situ and the subsequent analysis of the loss of function in 'knock-out' mice as a tool for basic research or in an attempt to mimic human diseases.
The in vitro micronucleus test as a tool to study resistance to compounds used in tumor therapy. A fast and reliable in vitro test to detect resistance of a tumor against therapeutic compounds before treatment would be of great value. These cell lines are known to exhibit resistancies to several therapeutic drugs with different mechanisms of activity to a different extend.
As model compounds for this investigation we used etoposide, mitoxantrone, daunoblastin, and idarubicin. We found a dose-dependent induction of effects in both systems at similar substance concentrations for each of the cell lines. However, the sensitivities for the compounds varied between the cell lines to a great degree 10 to fold. Of both assays, the micronucleus assay in general showed a response at lower substance concentrations than the cell growth assay. Thus, although much research in this area will be needed, it seems that the micronucleus test is a promising tool for the in vitro analysis of resistance to chemotherapeutic compounds that could be tested before therapy.
Modification of the HPRT-assay for photomutagenicity-testing. The photomutagenic potential of Bay Y , a photoreactive fluoroquinolone, was investigated in five protocol variations: Start of treatment with test substance and UV-irradiation at the same time standard protocol. Preincubation of the cells with test substance in the dark prior to irradiation. Using both protocols, Bay Y induced significant increases in mutation frequency over the concurrent irradiated vehicle controls.
Irradiation of cells and subsequent treatment with non-irradiated substance. Exposure of non-irradiated cells to test substance irradiated in culture medium. Reseeding of cells immediately after treatment and irradiation. No photomutagenic effects were evident using protocols Due to these results we conclude: The photomutagenic effect of Bay Y is not due to- a repair-inhibition of UV-induced DNA lesions - the formation of a stable photomutagenic product after UV-irradiation.
The question whether UV-induced radical formation is responsible for the effects seen in the standard protocol is currently under investigation using radical scavengers. The combined use of protocols 1 to 5 makes it possible to detect photomutagens with high sensitivity and to approach the mechanism of the photomutagenic effect. In the tester strain TA98 we found similar elevated reversion rates.
Testing with activated liver S9-fraction induced a slight additional increase. TA97a and TA showed no significant enhancement of spontaneous mutation rates. Hyperbaric oxygen therapy is clinically used for the treatment of a variety of diseases. Using the comet assay we have recently shown that HBO treatment under therapeutic conditions i. Therefore, HBO therapy is an excellent system for the investigation of DNA damage induced by oxidative stress in vivo.
ROS can attack DNA, thus producing distinctive patterns of DNA alterations including the oxidized base 8-oxoguanine which is a premutagenic lesion and seems to be involved in the induction of cancer. DNA damage was found directly after the treatment but could not be detected 24 h later.
Direct determination of 8-oxo-guanine by HPLC is now in progress to obtain information about the types of oxidative base damage induced by HBO treatment. As DNA damage was detected only after the first treatment but not after further treatments under the same conditions, an increase of antioxidative defences has been suggested.
We are now investigating changes in the antioxidant status i. To elucidate the biological significance of the DNA effects seen in the comet assay, micronuclei are evaluated to see if the observed DNA breakage is relevant for chromosomal damage. The AMES assay, a bacterial genotoxicity test generally required for registration, is a rapid and convenient method to get information on the mutagenic potential of a chemical.
For a full registration study up to 2 grams of test substance are needed. In early compound development, when often a first indication of the possible genotoxic potential is required, the available amount of substance may be limited.
To reduce substantially the required amount of test substance, a scaled-down version of the AMES test, the "AMES microsuspension assay" originally proposed by Kado et al. A number of known bacterial mutagens 4-nitroquinoline-N-oxide, mitomycin-C, 2-nitrofluorene, 9-aminoacridine, 2-aminoanthracene, cyclophosphamide were tested in both, the conventional AMES test and the AMES microsuspension assay.
In the latter 0. Thereafter this mixture was added to 2 ml of soft agar and plated on minimum agar plates. The plates were incubated and counted as in the conventional AMES test. For a full screening test 25 mg of test compound are needed. The results obtained indicate that the sensitivity of the microsuspension assay is at least that of the conventional AMES assay. The advantage of this screening system in comparison to others is that the same bacterial strains are used as in the normal AMES assay used for registration.
MSH2 mismatch repair activity of CHO cells is growth regulated and inversely related to resistance of cells to methylating agents. Mammalian cells aquire resistance to methylating mutagens and carcinogens by expression of the DNA repair protein alkyltransferase MGMT. We have generated another class of cells which are methylation resistant without expressing MGMT. The resistance pertained the end points cytotoxicity and chromosomal aberration formation.
These findings are taken to indicate that MSH2-dependent mismatch repair is growth regulated and can undergo quantitative changes which ultimately affect the level of methylating drug resistance.
The Ames-Test is one of a number of methods used to test the genotoxic potential of pollutants in surface waters. Because many surface water samples contain pollutants at trace levels, it is often necessary to concentrate the sample prior to testing. However, preconcentration techniques can in some cases induce chemical changes to the micropollutants within the sample.
Terahertz Electromagnetic Fields ( THz) Do Not Induce Manifest Genomic Damage In Vitro
gelelektrophorese-assay (comet assay) in A Zellen, in Hamster V79 als die der Kontrollsubstanzen: MMS (Methyl Methansulfonat; alkylierende Substanz). Zunächst werden Methoden zur Messung der Gentoxizität im Comet-Assay in after chronic exposure to methyl methanesulfonate in a multigeneration study. 27 Sep Cells were treated with µM methyl methanesulfonate (MMS) for 4 h. Both cell types exhibited significantly increased DNA damage as a.