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Biomedical subjects

R K Zahn

Publications and source records attributed to R K Zahn.

At least 19 recordsLinked to original sources
PubMed2001

Specific detection of cyclobutane pyrimidine dimers in phytoplankton DNA by a non-radioactive assay based on T4-endonuclease V digestion.

The effect of artificial and natural UV irradiation on DNA in marine phytoplankton Isochrysis galbana monoculture was investigated. The presence of cyclobutane pyrimidine dimers (CPDs) in unlabelled I. galbana DNA was detected by a non-radiometric alkaline filter elution assay after T4-endonuclease V digestion. The quantity of CPDs was estimated by alkaline agarose gel electrophoresis. Precise determination of the amount of DNA in the presence of I. galbana pigments was achieved by oxazole yellow homodimer (YOYO) dye. T4-endonuclease V-sensitive sites frequency (ESS/kb), measured after exposure to 2-40 kJ m(-2) of artificial UV light, increased in a dose-dependent manner. Twelve hours after irradiation cell culture growth was disrupted, and 50% of initial DNA damage in the cells was observed. After 1 h of sunlight exposure, the incidence of CPDs increase significantly. Prolonged exposition to sunlight decrease CPDs incidence due to efficiency of I. galbana DNA repair mechanisms. The presence of water-soluble crude oil fraction (WSOF) affected DNA repair efficiency resulting in accumulation of CPDs in I. galbana DNA.

Biological Assay↗
PubMed2000

DNA damage susceptibility and repair in correlation to calendric age and longevity.

In two mouse strains, SAM P (senescence acceleration prone) and SAM R (senescence acceleration resistant), of different longevities, with a ratio of P/R=1:2), the DNA status in the course of aging has been investigated using the DNA Alkaline Filter Elution (AFE) technique. Six different organs (brain, liver, heart, lung, intestine, and muscle) have been used in each of the four animals of a given age. Earlier it had been shown, that DNA is damaged the more the higher the age of the animal. DNA damage susceptibility, measured after exposure of organ pieces to nitroquinoline-N-oxide (NQO), is also significantly increased at higher ages, while repair, measured of NQO damaged tissue after 3 h incubation in full medium is significantly reduced. In the strain with shorter longevity the damage increments and the repair deficiencies are drastically deviating from those with higher longevity. These findings of strong coupling of the DNA status to aging as well as longevity suggest causative relations.

4-Nitroquinoline-1-oxide↗
PubMed2000

Age-associated DNA damage is accelerated in the senescence-accelerated mice.

We investigated how the DNA status correlates with the aging process in organisms, in different organs and in tissues using two inbred strains of mice, which are genetically related but have different senescence patterns. The SAMP1 mice belong to an accelerated senescence-prone and short lived strain, the other, SAMR1 mice are from an accelerated senescence-resistant and long lived strain. Using the alkaline filter elution technique, pieces of tissues from six organs: lung, intestine, liver, brain, muscle, and heart have been examined for DNA damage, mainly DNA single strand breaks. It was shown that in newborns the DNA damage is minimal, and it was increased significantly with calendric age in all organs in both strains. Although the correlation of DNA damage with aging differed in the different six organs, damage was significantly higher in SAMP1 mice than SAMR1 mice at later life in all organs. This is another remarkable example for the strong correlation of DNA damage and aging process, especially with senescence acceleration.

Aging↗
PubMed2000

Evaluation of the SOS/umu-test post-treatment assay for the detection of genotoxic activities of pure compounds and complex environmental mixtures.

This study presents an evaluation of the SOS/umu-test after introducing an additional dilution and incubation in the post-treatment assay. This treatment reduces the influence of coloured test compounds that otherwise affect the colorimetric determination of the beta-galactosidase activity and the bacterial growth measurement during the testing of complex environmental samples. The post-treatment assay significantly increased the beta-galactosidase activity and consequently the enzyme induction ratios at higher doses of model genotoxins 4-nitroquinoline-N-oxide, N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminoanthracene, benzo(a)pyrene with low or no effect on the sensitivity of the test itself. On the other hand tests of environmental extracts indicated significant increases in sensitivity after additional incubation. 4-Nitroquinoline-N-oxide treatments of bacteria in the test affected cell division and caused filamentous growth. The size of filamentous bacteria and incidence rate of the length categories was positively correlated with the concentrations of genotoxins. Presence of filamentous tester bacteria proved induction of SOS response and genotoxic activity of environment samples in SOS/umu-test.

4-Nitroquinoline-1-oxide↗
PubMed1999

A microplate assay for DNA damage determination (fast micromethod).

A rapid and convenient procedure for DNA damage determination in cell suspensions and solid tissues on single microplates was developed. The procedure is based on the ability of commercially available fluorochromes to interact preferentially with dsDNA in the presence of ssDNA, RNA, and proteins at high pH (>12.0), thus allowing direct measurements of DNA denaturation without sample handling or stepwise DNA separations. The method includes a simple and rapid 40-min sample lysis in the presence of EDTA, SDS, and high urea concentration at pH 10, followed by time-dependent DNA denaturation at pH 12.4 after NaOH addition. The time course and the extent of DNA denaturation is followed in a microplate fluorescence reader at room temperature for less than 1 h. The method requires only 30 ng DNA per single well and could conveniently be used whenever fast analysis of DNA integrity in small samples has to be done, e.g., in patients' lymphocytes after irradiation or chemotherapy (about 3000 cells per sample), in solid tissues or biopsies after homogenization (about 25 microg tissue per well), or in environmental samples for genotoxicity assessment.

Animals↗
PubMed1999

Assessment of DNA-protein crosslinks in the course of aging in two mouse strains by use of a modified alkaline filter elution applied to whole tissue samples.

Two different mouse strains have been used for determination of age dependence of DNA-protein crosslinks by alkaline filter elution: a long lived laboratory strain, NMRI and an accelerated senescence-prone, short lived strain, SAMP1. Five organs were selected: Brain, kidney, lung, heart and liver. Remarkably in all five organs of short lived SAMPI mice crosslinks increased significantly with age. In NMRI however only in brain and heart a significant rise in old age has been observed, while in the other organs there was no increase in DNA-protein crosslinking. Appreciable mitotic activity which is lacking in brain and heart could be the reason for this difference. Poor repair in all five organs could be an important component for the multiple ailments and shortened life span in SAMP1 mice.

Aging↗
PubMed1999

The influence of radiation and chemotherapy-related DNA strand breaks on carcinogenesis: an evaluation.

PURPOSE: DNA strand breaks are believed to induce carcinogenesis. This study was conducted to analyze induction and repair of irradiation- and chemotherapy-related strand breaks in vitro. METHODS: Friend Leukemia cells were exposed to irradiation and various chemotherapeutic agents at different doses and concentrations. Occurrence of strand breaks was determined fluorometrically, measuring the rate of DNA unwinding immediately after exposure and 24 hours later. RESULTS: The amount of double-stranded DNA decreased significantly for irradiation, doxorubicin, dactinomycin and etoposide (p < or = 0.05, t-test). After 24 hours free of exposure, the persistent damage was detectable for all of these agents but not for irradiated cells, with DNA strand breaks being decreased for etoposide, unchanged for doxorubicin and increased for methotrexate as well as for dactinomycin. CONCLUSIONS: Severe DNA damage is induced by various chemotherapeutic agents and by irradiation. While repair of chemotherapy-related strand breaks may remain incomplete or prolonged for some chemotherapeutic agents, repair of radiation induced strand breaks is faster and more complete. Therefore chemotherapy-related carcinogenesis may partially be explained by prolonged persistence of DNA strand breaks.

Animals↗
PubMed1997

Increase of sensitivity and validity of the SOS/umu-test after replacement of the beta-galactosidase reporter gene with luciferase.

The SOS/umu-test with Salmonella typhimurium TA1535/pSK1002 as tester strain is a rapid and valuable bacterial assay for screening of umuC-dependent mutagenic potential of chemical compounds and chemicals relevant to environmental pollution. The initial assay was modified by replacing the beta-galactosidase reporter gene with luciferase. Thereby, the sensitivity of the umu-test was increased significantly and the susceptibility to intensively coloured solutions was reduced. The alternative enzyme assay in the modified umu-test (umu-Luc) represents an independent method which allows to confirm the colorimetric results obtained with the original SOS/umu-test system (umu-Gal) by measuring the beta-galactosidase activity. It has been proved that for several genotoxins an increased induction rate could be achieved by applying the new umu-Luc test.

Blotting, Western↗
PubMed1996

DNA status in brain and heart as prominent co-determinant for life span? Assessing the different degrees of DNA damage, damage susceptibility, and repair capability in different organs of young and old mice.

Alkaline filter elution has been modified by a freeze-grinding step that allows the evaluation of the DNA status of whole tissue, including mouse tail cross-sections, with only small additional artefacts. Four to seven different organs from individually coordinated female NMRI mice, rated as young when 2-3 months, and as old when 24-27 months, of age, have been used. Tissues of individual mice differ significantly in their DNA status. Alkali-labile sites are relatively rare and differ in amount in the different organs in the young. They show significant increases in the old, reaching the highest values in the brain and the heart. Proteinase K dependent DNA-protein cross-links are not prominent nor are they increased with age in some organs, except in the brain and the heart. DNA damage susceptibility was measured after application of 3.5 microM nitoquinolin-N-oxide to 15 mg fresh. tissue pieces for 90 min. The susceptibility is large and varying in wide ranges in the different organs. Upon 3 h post-exposure incubation in full medium all samples showed DNA repair, the young reach nearly complete repair in the lung, while repair, generally, in the old is significantly decreased. In old brain and heart it is even near zero. This together with high values in alkali-labile sites and protein-DNA cross-linking suggests that these two organs may act as pacemakers and play a role as prominent co-determinants for the life span of the species.

Aging↗
PubMed1995

Increased genotoxicity of acetylaminofluorene by modulators of multixenobiotic resistance mechanism: studies with the fresh water clam Corbicula fluminea.

The presence of a 'multixenobiotic resistance' [MXR] mechanism in gills of the freshwater clam Corbicula fluminea was investigated. Western blot analyses of membrane vesicles from gills, applying antibodies to vertebrate P170 multidrug resistance (MDR) protein, revealed a 135 kDa immunoreactive protein. Verapamil caused a reduction of 3H-vincristine (3H-VCR) binding onto vesicles from clam. Exposure of clams to 3H-VCR in the presence of verapamil or staurosporine (STP) enhanced the accumulation of 3H-VCR over control values. Furthermore, clams were exposed instead to VCR, to a model carcinogen, 2-acetylaminofluorene (AAF), to determine the verapamil- and STP-dependent increase of single-strand breaks (SSBs) in DNA from gills of this organism. Verapamil caused no or little increase of SSBs induced by exposure to 0.01 or 0.10 microM AAF, respectively, as measured by the alkaline elution technique. In contrast, in the presence of STP a highly significant and dose-dependent enhancement of AAF-mediated SSBs was measured already at exposure to 0.01 microM AAF. These data indicate (i) that the clam C. fluminea is provided with a P-glycoprotein-like element of the MDR-mechanism, (ii) that this system can be poisoned by chemosensitizers such as verapamil and STP, (iii) the role of protein kinase C in the regulation of MXR function and (iv) the importance of the MXR modulators for the assessment of ecotoxicological effects of pollutants.

2-Acetylaminofluorene↗
PubMed1993

Nonradiometric detection of DNA crosslinks in mussel hemolymph by alkaline elution.

The use of bleomycin-Fe(II) complex in the determination of DNA crosslinks by alkaline elution was investigated for the purpose of developing a simple nonradiometric method applicable for detection of DNA damage in marine organisms. The assay involves preparation of active bleomycin-Fe(II) complex and its application in the washing step of the original alkaline elution procedure. Treatment of cell-free DNA from Friend leukemia cells and mussel hemolymph with bleomycin-Fe(II) complex directly on the filter resulted in a concentration-dependent strand scission response with excellent correlation (r = 0.94 and 0.98, respectively). The procedure was validated by determination of DNA cross-links in mussels injected with mitomycine C and formaldehyde.

Animals↗
PubMed1992

Fractionation of DNA from marine invertebrate (Maja crispata, Mytilus galloprovincialis) haemolymph by alkaline elution.

1. An alkaline elution procedure for the detection of DNA damage in marine invertebrate haemolymph has been developed. 2. Provided that three criteria are optimized, such as buffer composition, small filter pores (0.22 microns GVWP 025 00, Millipore), and optimal amounts of haemolymph applied, flow rates may be changed within the range of 0.2 ml/min to 0.05 ml/min without adverse back-pressure on the filter and without blocking filter pores. 3. Under optimal conditions, 70% of mussel haemolymph DNA, and 80% of crab haemolymph DNA will be retained on the filter after 6 hr of elution, indicating shorter DNA in mussel haemolymph. 4. The technique is applicable for testing the in vivo effects of different compounds on DNA in marine invertebrates, and to measurements of DNA damage in naturally exposed mussels. 5. This argues an important case for the use of alkaline elution technique for assessment of environmental genotoxicity, and especially for investigation of DNA damage in different marine organisms which cover a broad range in their DNA molecular weights.

Animals↗
PubMed1991

Genotoxicity of the fungicide dichlofluanid in seven assays.

Seven different endpoints for detection of genotoxicity have been used to demonstrate the DNA-altering properties of Dichlofluanid, a fungicide commonly used in viticulture pest control. Each endpoint (DNA synthesis inhibition test, alkaline viscosimetry, umu-test, alkaline filter elution, FADU-test, 32P-postlabeling, and electron microscopy) shows clear evidence of genotoxicity. These data indicate that application of the fungicide dichlofluanid may be mutagenic and/or carcinogenic for exposed humans.

Aniline Compounds↗
PubMed1991

A microplate version of the SOS/umu-test for rapid detection of genotoxins and genotoxic potentials of environmental samples.

The umu-microtest is a miniaturized automated short-term test version proposed for screening of umuC-dependent mutagenic potentials of chemicals relevant to environmental pollution, river water and industrial waste water. The test is based on the SOS/umu-test and has been modified in order to allow extensive testing of environmental samples. Genetically engineered Salmonella typhimurium (TA1535/pSK1002) are incubated on a microplate rotor in a sloping position for 2 h with the test samples, followed by addition of fresh culture medium to reach a 10-fold dilution of the incubation medium. 2 h later, the activity of the beta-galactosidase, which reflects umuC induction, is determined colorimetrically. The incubation of the bacteria in the presence of the test compounds as well as the assessment of beta-galactosidase activity takes place in 96-well microplates, thus enabling simultaneous screening of large numbers of samples. Data of the genotoxic potentials are available within 8 h. Computer-controlled automation is possible by using a laboratory workstation.

Azides↗
PubMed1990

A microplate version of the DNA-synthesis inhibition test for rapid detection of DNA-alteration potentials.

A microplate version of the DNA-synthesis inhibition test (DIT) for fast detection of DNA-alteration potentials has been developed. The DIT is based on the concept that DNA damage causes inhibition of DNA synthesis that becomes detectable some time after replicating cells have been in contact with genotoxic agents. In this test procedure human tissue culture cells (HeLa S3), prelabeled with [14C]thymidine, arfe exposed for 90 min to the substances in question. After the cells are rinsed, they are allowed to recover for 2 1/2 h in fresh culture medium, thereby unspecific interactions interfering with DNA replication are practically eliminated. Next, [3H]thymidine is added for 30 min, and then the cells are harvested and thoroughly rinsed. Finally, incorporated radioactivity is determined by liquid scintillation counting for measurement of the 3H/14C ratio. This allows for the evaluation of DNA synthesis during the 3H-labeling period and of the extent of genotoxic damage. This microplate version of the DIT can be carried out fully automated in a laboratory workstation. The test is compared to other tests for genotoxicity. Its advantages are discussed.

Autoanalysis↗
PubMed1990

Alternating current voltammetric determination of DNA damage.

The conditions for alternating current (a.c.) voltammetric DNA determinations have been investigated with respect to its use with alkaline filter elution techniques at low DNA concentrations. In inorganic electrolyte solutions three current peaks can be distinguished: peak I around -1.1 V caused by the reorientation or desorption of DNA segments; peak II around -1.2 V caused by the native DNA (nDNA) form; peak III caused by denatured DNA (dDNA) at -1.4 V. Sonication of nDNA increases the peak current, however not with dDNA. Both dDNA and nDNA give linear peak current increments with DNA increments, their regression lines cutting the concentration axis at the origin. In filter elution techniques organic bases are often used. Adding ethanolamine (EA) elution buffer decreases the peak amplitude of DNA. It turns out that an unknown substance, perhaps a protein or RNA, elutes from the filters and gives rise to a current peak at about -1.3 V. This substance can interfere with the dDNA by competing for electrode surface area, since it diffuses much faster than the large molecules of the DNA. Since however, dDNA has a higher affinity for the electrode surface, after enough time, usually few minutes, the dDNA increasingly displaces the substance and occupies the surface. The same is valid for other organic molecules and thus also for EA. It is therefore remarkable that the unknown substance can be altered by ultrasonication, so that it will no longer interfere with dDNA, in contrast to EA. EA, on the other hand, can be "titrated". When EA is present at short accumulation times it prevents dDNA adsorption. By adding dDNA, the EA can be scavanged and further addition will adsorb and thus increase peak current in proportion to the concentration of the DNA present. The conditions for voltammetric DNA determination have been investigated obeying the recognized interactions. Avoiding organic bases and using inorganic ones would simplify the determination procedure. The reproducibility of the procedure in the range of 50-60 ng DNA/ml has been found to be +/- 6%.

Animals↗
PubMed1990

Application of the SOS umu-test in detection of pollution using fish liver S9 fraction.

1. The possibility of Aroclor 1254 and beta-naphthoflavone treated fish Mugil auratus and fish sampled in low and high polluted areas to convert some premutagens to active intermediers in the SOS umu-test have been investigated. 2. Genotoxicity of Aflatoxin b1 differed markedly upon activation with liver S9 fractions from fish with different pollution histories, with the highest activation potency in fish living near a fish cannery. 3. Inhibition of umu gene expression by 7,8-benzoflavone in vitro clearly demonstrates a cytochrome P-450 mediated activation of aflatoxin b1. 4. 2-Aminoanthracene and 2-aminofluorene were weakly activated to genotoxic products and the induction of umu gene expression could be detected only in the presence of S9 fractions from fish treated with beta-naphthoflavone and Aroclor 1254 in the laboratory. 5. The capability of S9 from fish living near a fish cannery to convert 2-aminoanthracene and 2-aminofluorene was not observed.

Animals↗