Search PubMed⌕ Search

Biomedical subjects

E Mullaart

Publications and source records attributed to E Mullaart.

At least 37 records · Page 2Linked to original sources
PubMed1989

Age-related induction and disappearance of carcinogen-DNA-adducts in livers of rats exposed to low levels of 2-acetylaminofluorene.

It was investigated whether in vivo aging of rat liver is associated with changes in the induction and rate of disappearance of DNA damage. For this purpose 6- and 36-month-old rats were intraperitoneally injected with a single, low dose (5 mg/kg body wt.) of the model liver carcinogen 2-acetylaminofluorene (AAF). Using the 32P-postlabeling assay we found that N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) was the major DNA-adduct formed. The minor adduct N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) could only be detected after doses of 20 mg/kg or more. Quantitation of adduct levels at various time points after treatment indicated a rapid induction of AF-adducts, which were already present at 6 h after treatment. The subsequent loss of AF-adducts was relatively slow, as was indicated by the presence of a substantial amount of AF-adducts as late as 21 days after treatment. Slight age-related differences in the pattern of induction and disappearance of AF-adducts and a somewhat higher level of persisting lesions in old than in young rats were observed.

2-Acetylaminofluorene↗
PubMed1989

Quiescent human peripheral blood lymphocytes do not contain a sizable amount of preexistent DNA single-strand breaks.

Sedimentation of nucleoids through neutral sucrose density gradients has shown that nucleoids isolated from phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBL) sediment faster than nucleoids derived from quiescent lymphocytes, which was attributed to rejoining of DNA single-strand breaks (SSB) present in the resting cells (A.P. Johnstone, and G.T. Williams (1982) Nature (London) 300, 368). We isolated PBL from donors and determined the amount of SSB in nonradiolabeled, untreated resting and PHA-stimulated cells by applying the alkaline filter elution technique. Calibration was based on dose-dependent induction of SSB by 60Co-gamma-radiation. Quiescent cells did not contain a sizable amount of SSB. Mitogen-stimulated cells showed equally low amounts of SSB per cell. The present study indicates that the interpretation of the results obtained with the nucleoid sedimentation technique concerning the supposed rejoining of SSB in PHA-stimulated human lymphocytes is incorrect. Other, equally sensitive, techniques such as alkaline filter elution appear to be preferable for studies on DNA damage and repair.

DNA Damage↗
PubMed1989

Effects of lethal exposure to hyperoxia and to hydrogen peroxide on NAD(H) and ATP pools in Chinese hamster ovary cells.

Cell death by oxidative stress has been proposed to be based on suicidal NAD depletion, typically followed by ATP depletion, caused by the NAD-consuming enzyme poly(ADP)ribose polymerase, which becomes activated by the presence of excessive DNA-strand breaks. In this study NAD+, NADH and ATP levels as well as DNA-strand breaks (assayed by alkaline elution) were determined in Chinese hamster ovary (CHO) cells treated with either H2O2 or hyperoxia to a level of more than 80% clonogenic cell killing. With H2O2 extensive DNA damage and NAD depletion were observed, while at a higher H2O2 dosage ATP also became depleted. In agreement with results of others, the poly(ADP)ribose polymerase inhibitor 3-aminobenzamide completely prevented NAD depletion. However, both H2O2-induced ATP depletion and cell killing were unaffected by the inhibitor, suggesting that ATP depletion may be a more critical factor than NAD depletion in H2O2-induced killing of CHO cells. With hyperoxia, only moderate DNA damage (2 X background) and no NAD depletion were observed, whereas ATP became largely (70%) depleted. We conclude that (1) there is no direct relation between ATP and NAD depletion in CHO cells subjected to toxic doses of H2O2 or hyperoxia; (2) H2O2-induced NAD depletion is not by itself sufficient to kill CHO cells; (3) killing of CHO cells by hyperoxia is not due to NAD depletion, but may be due to depletion of ATP.

Adenosine Triphosphate↗
PubMed1989

The removal of UV-induced pyrimidine dimers from DNA of rat skin cells in vitro and in vivo in relation to aging.

Young and old rats were compared with respect to the capacity of their skin fibroblasts and epidermal keratinocytes to remove low levels of ultraviolet light (UV) induced UV-endonuclease sensitive sites (pyrimidime dimers) from their DNA, in vitro and in vivo, respectively. In vitro, over a 24-h time period, fibroblasts from both young and old rats were found to remove about 20% of the pyrimidine dimers originally induced by 4.6 J/m2 of UV-C. In vivo, after 2.6 kJ/m2 of UV-B hardly any UV lesions were found to be present in fibroblasts, as demonstrated by immunohistochemistry using an anti-thymine dimer antibody. As reported earlier (Mullaart et al., J. Invest. Dermatol., 90 (1988) 346-349) cultured epidermal keratinocytes do not differ from cultured fibroblasts in UV repair kinetics, whereas in vivo they remove at least 50% of the pyrimidine dimers induced by 4 kJ/m2 of UV-B within 3 h. We now show that epidermal keratinocytes from old rats are not deficient in their in vivo repair characteristics upon this low UV-B dose. However, since a considerable fraction of the pyrimidine dimers appeared to be persistent in fibroblasts and keratinocytes, demonstrated by both enzymatic and immunochemical assays, the possibility is discussed that long-term exposure of skin cells to UV may lead to an accumulation of DNA damage with age.

Aging↗
PubMed1989

Chromosomal instability in an oxygen-tolerant variant of Chinese hamster ovary cells.

Background levels of chromosomal aberrations and sister-chromatid exchanges (SCEs) were determined in CHO-99 cells, an oxygen-tolerant variant substrain of Chinese hamster ovary (CHO-20) cells capable of stable proliferation under an atmosphere of 99% O2/1% CO2, a level of hyperoxia at which cultured mammalian cells normally cannot survive. The mean chromosomal aberration frequency in CHO-99 cells was as high as 1 aberration per cell (mainly chromatid and chromosome gaps and breaks) versus 0.05 aberration/cell in CHO-20 cells, while the SCE frequency was 1.7- to 2.1-fold increased. While most aberrations were apparently distributed at random over the chromosomes, up to 31% of the aberrations appeared to be involved in site-specific fragility at a homologous site in chromosomes Z3 and Z4. Immediately upon shifting CHO-99 cells to air-equilibrated conditions their SCE frequency decreased to the control level, whereas the aberration rate persisted at a still elevated level of 0.16-0.31 aberration per cell, even after a culture period of 14 weeks under normoxia. This indicates that at least part of the chromosomal instability is a constitutional property of the variant cells, i.e., not directly dependent upon hyperoxic stress. In CHO-99 X CHO-20 hybrids the occurrence of chromatid-type aberrations and fragile site but not that of chromosome-type aberrations was suppressed under normoxic conditions, suggesting that chromatid-type aberrations and fragile site expression on the one hand and chromosome-type aberrations on the other hand are mediated by different constitutional defects in CHO-99 cells. No gross alterations in (deoxy)ribonucleoside triphosphate pools were detected in CHO-99 cells that could be held responsible for their chromosomal instability. In addition, no increased level of DNA damage was detected by the technique of alkaline elution. The excessive chromosomal instability in CHO-99 cells, as observed under hyperoxic conditions, may originate from reactive intermediates giving rise to DNA double-strand breaks and/or a type of DNA lesion that is resistant to the conditions of the alkaline elution technique. However, alternative mechanisms based upon reactive species interfering with DNA replication/repair processes cannot be excluded.

Animals↗
PubMed1989

Genotoxic effects of intragastrically administered benzo[a]pyrene in rat liver and intestinal cells.

The genotoxic effects of intragastrically administered benzo[a]pyrene (BaP) were studied in isolated rat liver and intestinal cells. We found that BaP was unable to induce unscheduled DNA synthesis in rat parenchymal liver cells in vivo. In contrast, alkaline elution showed that at 5 h after administration of BaP a considerable number of alkali-labile sites was present in the DNA of both intestinal cells and parenchymal liver cells, but not in that of non-parenchymal liver cells. The possibility is discussed that these sites were induced by radical intermediates, generated during the metabolization of BaP.

Animals↗
PubMed1988

Age-dependent accumulation of alkali-labile sites in DNA of post-mitotic but not in that of mitotic rat liver cells.

The amount of spontaneous damage in the DNA of rat liver cells was measured by using the alkaline elution assay. An age-related increase of approximately 700 detectable alkali-labile sites (80%) was found for rat parenchymal liver cells; cells from 6-month-old rats contained approximately 900 alkali-labile sites per cell while cells from 36-month-old rats contained approximately 1600 alkali-labile sites. In contrast to the situation with the postmitotic parenchymal liver cells, no age-related increase in the number of alkali-labile sites was found for the non-parenchymal liver cell fraction, which has a higher mitotic activity. These results support the hypothesis that aging takes place predominantly in postmitotic cells.

Aging↗
PubMed1988

Differences in pyrimidine dimer removal between rat skin cells in vitro and in vivo.

Pyrimidine dimers, the most abundant type of DNA lesions induced by ultraviolet light (UV), are rapidly repaired in human skin fibroblasts in vitro. In the same cell type from rats, however, there is hardly any removal of such dimers. To investigate whether this low capacity of rat skin cells to repair lesions in their DNA is an inherent characteristic of this species or an artifact due to cell culturing, we measured the removal of UV-induced pyrimidine dimers from rat epidermal keratinocytes both in vitro and in vivo. Epidermal keratinocytes in vitro were unable to remove any dimers over the first 3 h after UV-irradiation, while only about 20% was removed during a repair period of 24 h. In this respect, these cells were not different from cultured rat fibroblasts. In contrast to the results obtained with keratinocytes in vitro, we observed a rapid repair of pyrimidine dimers in UV-irradiated keratinocytes in vivo over the first 3 h; this rapid repair phase was followed by a much slower repair phase between 3 and 24 h. These results are discussed in terms of the possibility that mammalian cells are able to switch from one DNA repair pathway to another.

Animals↗
PubMed1986

Immunochemical detection of DNA in alkaline sucrose gradient fractions.

A procedure for the detection of non-radioactive DNA in alkaline sucrose gradients is described. This method consists of the following steps: (1) fractionation of the DNA-containing gradients into microtiter plates, neutralization and overnight adsorption; (2) covalent labelling of the guanine bases of the adsorbed DNA by reaction with N-acetoxy-2-acetylaminofluorene; and (3) determination of the gradient profile by means of an enzyme-linked immunosorbent assay using antibodies with high affinity for dG-AAF. The method has been found suitable for the rapid and sensitive determination of gamma-ray-induced single-strand breaks and UV-induced pyrimidine dimers in mammalian cells in vitro. In addition, it has been shown that the method can be used for the determination of pyrimidine dimers induced in skin cells in vivo.

Acetoxyacetylaminofluorene↗
PubMed1986

UV-induced DNA excision repair in rat fibroblasts during immortalization and terminal differentiation in vitro.

UV-induced DNA excision repair was studied as DNA repair synthesis and dimer removal in rat fibroblast cultures, initiated from either dense or sparse inocula of primary cells grown from skin biopsies. During passaging in vitro an initial increase in DNA repair synthesis, determined both autoradiographically as unscheduled DNA synthesis (UDS) and by means of the BrdU photolysis assay as the number and average size of repair patches, was found to be associated with a morphological shift from small spindle-shaped to large pleiomorphic cells observed over the first twenty generations. In cell populations in growth crisis, a situation exclusively associated with thin-inoculum cultures in which the population predominantly consisted of large pleiomorphic cells, UDS was found to occur at a low level. After development of secondary cultures into immortal cell lines, both repair synthesis and morphology appeared to be the same as in the original primary spindle-shaped cells. At all passages the capacity to remove UV-induced pyrimidine dimers was found to be low, as indicated by the persistence of Micrococcus luteus UV endonuclease-sensitive sites. These results are discussed in the context of terminal differentiation and immortalization of rat fibroblasts upon establishment in vitro.

Animals↗
PubMed1985

UV-induced unscheduled DNA synthesis in fibroblasts of aging inbred rats.

Because of the suggested relationship between the lifespan of an organism and the amount of unscheduled DNA synthesis (UDS) occurring in its cells after treatment with genotoxic agents, we initiated a lifespan study of this step of the nucleotide excision repair pathway in female Wistar (WAG/Rij) rats. Skin fibroblasts were isolated at 2 time points, separated by a 9-month interval, from rats of various ages. The isolated cells were cultured for 1 passage, irradiated with ultraviolet light (UV) and analyzed by autoradiography for their capacity to perform UDS. The results of the two cross-sectional series of determinations were identical: small variations among individual animals and a slight, but statistically significant age-related decrease in the initial rate but not in the end level of UV-induced UDS. The small variation among individual inbred rats as compared with the large variation reported for UDS in human populations suggests that the latter is largely due to genetic differences. The lack of a more pronounced age-related decrease along with the small individual variation suggests that the activity of the DNA nucleotide excision repair pathway is not an important single determinant of individual longevity in inbred rats of the same strain and sex.

Aging↗
PubMed1982

A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma.

An indirect spectrophotometric assay for extrinsic plasminogen activator has been devised, which is based on the parabolic assay of Drapier et al. (5). The system contains activator, plasminogen, the synthetic plasmin substrate H-D-Val-Leu-Lys-pNA (S-2251, Kabi) and a mixture of soluble fibrinogen fragments prepared by treatment of fibrinogen with cyanogen bromide. The addition of these fibrinogen fragments considerably enhances the sensitivity and specificity of the method owing to specific stimulation of the plasminogen activation by extrinsic plasminogen activator. The assay conditions were optimized and the application for extrinsic plasminogen activator measurements in plasma euglobulin fractions is demonstrated.

Fibrin↗
PubMedDate not supplied

Genome scanning by two-dimensional DNA typing: the use of repetitive DNA sequences for rapid mapping of genetic traits.

The existence of repetitive DNA sequences offers the possibility to assess the mammalian genome for individual variation in its entirety rather than at one or only a few sites. In order to fully explore the various sets of mammalian repeat sequences for this purpose, analytical tools are required which allow many if not all individual members of sets of repetitive elements to be resolved and identified in terms of location and allelic variation. We have applied and further developed an electrophoretic system, two-dimensional DNA typing, which may fulfill these requirements. The two-dimensional system combines separation of DNA fragments by size in a neutral gel, with separation by sequence composition in a denaturing gradient gel. By hybridization with minisatellite- and simple-sequence core probes and by inter-repeat polymerase chain reaction techniques, it is possible to obtain individual--and even chromosome-specific separation patterns that consist of hundreds of spots. Computerized image analysis and matching of such spot patterns allows the rapid assessment of multiple polymorphisms, spread over the genome, to monitor genetic variability in populations. When coupled to databases of polymorphic DNA markers with a known genomic location, two-dimensional DNA typing can greatly accelerate the mapping of genetic traits in humans, animals, and plants.

Alleles↗
PubMedDate not supplied

Kinetics of ultraviolet induced DNA excision repair in rat and human fibroblasts.

To obtain more information on the well-documented low excision-repair capacity of rodent cells in comparison with human cells, we have studied this form of DNA repair in UV-irradiated human and rat skin fibroblasts. For this purpose, we have determined (i) unscheduled DNA synthesis (UDS), using autoradiography, (ii) the number and size of repaired sites with the bromodeoxyuridine (BrdU) photolysis assay and (iii) the removal of Micrococcus luteus UV-endonuclease susceptible sites (ESS). We found rat cells to be quite capable of performing DNA-repair synthesis, as demonstrated by both UDS and BrdU photolysis, whereas they almost completely lacked the capacity to remove pyrimidine dimers, as indicated by the persistence of ESS. This discrepancy will be discussed in terms of the types of mechanisms by which mammalian cells may recognize and remove UV-induced photoproducts.

Animals↗
PubMedDate not supplied

Increased levels of DNA breaks in cerebral cortex of Alzheimer's disease patients.

It has been hypothesized that Alzheimer's disease (AD) is caused by an accumulation of damage in DNA due to defective DNA-repair (21). Attempts to test this hypothesis by determining the activity of DNA-repair systems in nonneuronal cells from AD patients and controls so far provided conflicting results. An alternative approach is the direct comparison of DNA-damage levels in neuronal tissue of AD patients and controls. In the present study we assayed the level of DNA breaks and alkali-labile sites in cerebral cortex tissue samples from AD patients and controls obtained from rapid autopsies. Our data on 11 AD patients and 8 control subjects indicate an at least two-fold higher level of DNA damage in cortex of AD patients as compared to controls.

Aged↗
PubMedDate not supplied

Decreased DNA repair capacity in familial, but not in sporadic Alzheimer's disease.

Using the alkaline filter elution technique we determined the induction and disappearance of DNA single-strand breaks (SSB) in freshly isolated peripheral blood lymphocytes (PBL) from 43 Alzheimer's disease (AD) patients and 48 normal, healthy age- and sex-matched control subjects following in vitro exposure to N-ethyl-N-nitrosourea (ENU). The mean percentage SSB disappearance in PBL from control subjects at 1 h after ENU treatment was 41.4 +/- 2.9%; this was not significantly different from that found in samples from AD patients which had no (n = 16) or one (n = 12) first-degree relative with dementia (42.5 +/- 8.2% and 43.0 +/- 4.4%, respectively; p greater than 0.75). However, in PBL of 15 AD patients with at least two first-degree relatives with dementia the mean percentage SSB disappearance was 23.6 +/- 5.8%, which was significantly lower than that found in controls (p less than 0.01) or in the other AD patients (p less than 0.02).

Aged↗
PubMedDate not supplied

DNA damage metabolism and aging.

As a result of permanent exposure to low levels of various endogenous and exogenous genotoxic agents, large numbers of lesions are continuously induced in the DNA of cells of living organisms. Such lesions could lead to dysfunction of cells and tissues, and they might well be the underlying cause of the age-related reduction of homeostatic capacity and the increased incidence of cancer and other diseases of old age. The rate of damage induction as well as the persistence of the lesions depends on the activity, efficiency and reliability of a wide variety of molecular defense systems. However, a certain degree of imperfection seems to be a general characteristic of most of these defense systems and this could lead to a gradual accumulation of DNA alterations during aging. Even when the original lesions are quickly removed, they can still lead to secondary changes in the DNA, such as DNA-sequence changes and changes in gene expression. This process would be accelerated in case of the occurrence of an age-related decline in the efficiency of these molecular defense systems. This review deals with the present knowledge on the occurrence of 'spontaneous' DNA damage in aging organisms, its potential sources, the influence of preventive and processive cellular defense mechanisms and its consequences in terms of DNA-sequence changes, DNA conformational and configurational changes and changes in gene expression. In general, it can be concluded from the data discussed here that, in spite of a number of discrepancies and conflicting results, an age-related accumulation of DNA alterations occurs at all levels, e.g., chemical structure, DNA-sequence organization and gene expression.

Aging↗