DNA Unwinding Technique Research Articles

Contaminant effects in shore crabs (Carcinus maenas) from Ria Formosa Lagoon

Defence and damage biomarkers signals were studied in female and male shore crabs Carcinus maenas transplanted between two sites at Ria Formosa Lagoon (South of Portugal). The cross transplantation occurred during 6 days at a hypothetical reference site (Ramalhete), and a contaminated site (Olhao). DNA unwinding technique was used for DNA integrity measurement. General enzymatic antioxidant responses in gills and hepatopancreas (catalase and gills glutathione peroxidase activities increase) showed that cross transplanted crabs (female and male) are suffering from pro-oxidant challenges at the Olhao site. Gills and hepatopancreas glutathione-S-transferase were reduced in both gender crabs transplanted from Ramalhete to Olhao. Metallothioneins induction occurred in crabs transplanted from Ramalhete to Olhao (contaminated by metals and polycyclic aromatic hydrocarbons (PAHs)). However metallothionein differences were also observed towards gender and organ specificities. High gills lipid peroxidation exist in male crabs transplanted from Ramalhete to Olhao, while in females it was the opposite. In both gender crabs from the Olhao site, gills DNA integrity decreased compared to the Ramalhete feral crabs. Moreover, hepatopancreas DNA integrity decreased in male crabs transplanted from Olhao to Ramalhete site which may be related to the environmental conditions (lower contamination levels) revealing the difficulty of selection of reference sites in field studies. Data demonstrated that female and male C. maenas antioxidant defences and damage biomarkers were sensitive to the mixture of contaminants present in these sites as well as good indicators of general stress.

Contaminant effects in shore crabs ( Carcinus maenas) from Ria Formosa Lagoon

Defence and damage biomarkers signals were studied in female and male shore crabs Carcinus maenas transplanted between two sites at Ria Formosa Lagoon (South of Portugal). The cross transplantation occurred during 6 days at a hypothetical reference site (Ramalhete), and a contaminated site (Olhão). DNA unwinding technique was used for DNA integrity measurement. General enzymatic antioxidant responses in gills and hepatopancreas (catalase and gills glutathione peroxidase activities increase) showed that cross transplanted crabs (female and male) are suffering from pro-oxidant challenges at the Olhão site. Gills and hepatopancreas glutathione-S-transferase were reduced in both gender crabs transplanted from Ramalhete to Olhão. Metallothioneins induction occurred in crabs transplanted from Ramalhete to Olhão (contaminated by metals and polycyclic aromatic hydrocarbons (PAHs)). However metallothionein differences were also observed towards gender and organ specificities. High gills lipid peroxidation exist in male crabs transplanted from Ramalhete to Olhão, while in females it was the opposite. In both gender crabs from the Olhão site, gills DNA integrity decreased compared to the Ramalhete feral crabs. Moreover, hepatopancreas DNA integrity decreased in male crabs transplanted from Olhão to Ramalhete site which may be related to the environmental conditions (lower contamination levels) revealing the difficulty of selection of reference sites in field studies. Data demonstrated that female and male C. maenas antioxidant defences and damage biomarkers were sensitive to the mixture of contaminants present in these sites as well as good indicators of general stress.

Protective action of copper (II) complex of a Schiff base against DNA damage induced by m-chloroperbenzoic acid using a novel DNA unwinding technique

DNA strand breaks can be detected with great sensitivity by exposing calf thymus DNA to alkaline solutions and monitoring the rate of strand unwinding. Fluorometric analysis of DNA unwinding (FADU) is a reliable method for detecting single-strand DNA breaks as an index of DNA damage induced by photosensitizer. m-Chloroperbenzoic acid (CPBA) was used as a photosensitizer in the photodamage of calf thymus DNA. When DNA is exposed to ionizing radiation, the radicals produced in the irradiated sample modify the base-pair regions of the double strands. The protective action of copper salt, Schiff base [ethylene diamine with ethyl acetate](L) and its Cu(II) complex (Cu 7 L Cl 14) against DNA damage photoinduced by CPBA was studied using ethidium bromide as a fluorescent probe. Treatment of DNA with 5, 10, 50, 100, or 200 μM CPBA produced 75%, 48%, 38%, 32% and 30% double-stranded DNA remaining, respectively after 30 min of alkaline treatment at 15 °C. Treatment of calf thymus DNA irradiated with CPBA with a dose of 1 mM [Cu 7 L Cl 14] produced 96% double-stranded remaining protection under the same conditions compared with irradiated DNA without addition of Cu(II) complex of Schiff base.

Peripheral blood lymphocytes DNA in patients with chronic liver diseases.

Viral replication in blood cells with nucleases may lead to the damage of lymphocytes genetic apparatus and the beginning of immunopathological reactions. Of this investigation is to reveal the damage to peripheral blood lymphocytes (PBL) DNA in the patients with chronic liver diseases. Sixteen-nine patients with chronic liver diseases (37 patients with chronic viral hepatitis, 2 patients with liver cirrhosis of mixed etiology (alcohol+virus G), 30 women with primary biliary cirrhosis-PBC) were examined. The condition of DNA structure of PBL was measured by the fluorescence analysis of DNA unwinding (FADU) technique with modification. Changes of fluorescence (in %) reflected the DNA distractions degree (the presence of DNA single-stranded breaks and alkalinelabile sights). The quantity of DNA single-stranded breaks and alkalinelabile sights in DNA in all patients with chronic viral hepatitis didn't differ from the control group, excluding the patients with chronic hepatitis (CH) C+G. Patients with HGV and TTV monoinfection had demonstrated the increase of the DNA single-stranded breaks PBL quantity. This fact may be connected with hypothesis about the viruses replication in white blood cells discussed in the literature. Tendency to increase quantity of DNA PBL damages in the patients with primary biliary cirrhosis (PBC) accordingly to the alkaline phosphatase activity increase was revealed. Significant decrease of the DNA single-stranded breaks and alkalinelabile sights in the PBC patients that were treated with prednisone was demonstrated. Probably, the tendency to increase the quantity of DNA single stranded breaks and alkalinelabile sights in lymphocytes of the PBC patients was depended on the surplus of the blood bile acid content.

Etching and Damage Action on Microbes' Cells by Low Energy N+ Beam

The action of etching and damage by 20 keV N+ beam on the cells of Deinococcus radiodurans and Escherichia coli was investigated by scanning electron microscope (SEM) and the electron spin resonance (ESR) spectrum of free radicals. The results showed that N+ implantation exerted the direct action of etching and damage of momentum transferring and the indirect action of the free radicals of energy deposition on their cells, many microholes were found on the surface of cells' wall and/or membrane by SEM, the damaged DNA was determined using DNA unwinding technique, and the signal of free radicals was measured by ESR. The degree of damage to cells by ion beam gradually increased with the increase implantation dose. With the post-treatment of 2 mmol/l caffeine and 0.5 mmol/l Na2-EDTA, the survival rate of D.radiodurans and E.coli further decreased in the order of caffeine > Na2-EDTA > control, and this suggested that low energy ion beam could be implanted into nucleus, doing a damage to DNA and resulting in the mutation of organisms.

Topoisomerase II-mediated alterations of K562 drug resistant sublines.

In order to further elucidate the roles of DNA topoisomerase II (topo II) subtypes, alpha and beta, as drug targets in chemotherapy, we have determined the enzyme levels in K562 cells selected for resistance to mitoxantrone (K562/Mxn), daunorubicin (K562/Dnr) and idarubicin (K562/Ida 20 and K562/Ida 60), as well as topo II-DNA complex formation, DNA damage and cytotoxicity, induced by topo II interactive agents, for example etoposide, teniposide, mitoxantrone and amsacrine. As compared to the parental cells, topo IIalpha/beta protein levels in K562/Mxn, K562/Dnr, K562/Ida 20 and 60 lines, measured with Western blot, were 17/67%, 85/88, 24/31% and 10/7% respectively. DNA damage, determined by DNA unwinding technique, induced by teniposide and amsacrine correlated with both topo IIalpha/beta protein levels (r2 = 0.8/0.9, P = 0.03/0.01 and r2 = 0.8/0.9, P = 0.04/0.01, respectively). Topo II-DNA complex formation induced by all studied drugs correlated with topo IIbeta protein levels (r2-range 0.8-0.9, P-range 0.01-0.04), while the correlation with topo IIalpha was weaker. Topo IIalpha/beta protein levels tended to show an inverse correlation with the cytotoxicity of etoposide (r2 = -0.9/-0.7, P = 0.01/0.06). The overall topo II-DNA complex formation correlated with drug-induced DNA damage (r2 = 0.9, P = 0.0001), whilst not with the cytotoxicity. Our findings indicate that both topo II isozymes are the targets of the antitumor agents studied, and of potential clinical relevance for prediction of treatment efficacy. They could play a role in tailored chemotherapy.

Etoposide‐induced DNA strand breaks in relation to p‐glycoprotein and topoisomerase II protein expression in leukaemic cells from patients with AML and CLL

Elevated expression of the membrane transporter p-glycoprotein (pgp) and impaired expression of the nuclear enzyme topoisomerase II (topo II) are well-known mechanisms for in vitro acquired drug resistance. The clinical relevance of topo II remains unclear, whereas a relationship between pgp levels and treatment results has been shown in acute myelogenous leukaemia (AML). We have investigated the relationships between the levels of topo II and pgp, and in vitro sensitivity to etoposide in mononuclear blood cells from 24 patients with AML, 16 with chronic lymphocytic leukaemia (CLL) and five healthy blood donors. Following incubation with etoposide, AML cells showed more DNA damage, determined by a DNA unwinding technique, than CLL cells (P = 0.001), whereas there was no difference in cellular etoposide accumulation. Pgp and topo IIbeta levels, determined by Western blot, showed a pronounced variation between patients, but no correlation with induced DNA damage, whereas topo IIalpha protein was undetectable. In the AML group, topo IIbeta expression correlated with pgp expression (rho = 0.7, P = 0.001, n = 24). The topo IIbeta expression was 147.4(+/-74.6)% in the pgp+ AML cells (n = 10), compared to 33.4(+/-27.8)% in pgp- AML cells (n = 14) (P = 0.0001). Our results show a previously unknown coexpression of topo IIbeta and pgp in AML, thereby suggesting that topo IIbeta is a potentially interesting resistance factor in AML.

Effects of ageing on nuclear DNA integrity and metabolism in mussel cells (Mytilus edulis L.)

As the age of a cell increases, so does the potential for DNA damage. Recent theories on ageing suggest accumulative DNA damage is the primary cause of cellular senescence, possibly due to the decreased ability of DNA to act as a template for gene expression.In this paper we investigate the effects of ageing on the level of nuclear DNA damage in tissues of wild mussels of three different age groups; 2–4 years (group I), 6–8 years (group II) and 10 years (group III). In the digestive gland and haemolymph cells, a significant age-dependent increase of DNA damage was observed, as evaluated by the fluorimetric alkaline DNA unwinding technique, which is able to detect both direct single strand DNA breaks as well as alkali-labile apurinic sites.In addition, the rate of DNA polymerase activity was studied in order to determine whether DNA damage was dependent on DNA alteration, or because of a reduced rate of DNA repair. Unscheduled DNA repair synthesis in isolated nuclei of digestive gland cells in older mussels, was significantly decreased in comparison to younger mussels (−42% in group II and −37% in group III, p<0.01). In the digestive gland, salt extraction gives a slight, but significant, decrease of aphidicolin-sensitive DNA polymerase activity in age group III of −25%, p<0.05.Finally, we looked at the age variation in relation to oxidative stress. This was evaluated by measuring malondialdehyde accumulation in mussel cells. Digestive gland cells of group III, showed a significant age-related increase in malondialdehyde content of 170%, p<0.01, indicative of enhanced peroxidative processes.Taken together, these data suggest that the accumulation of DNA damage in group II is mainly dependent on the impairment of DNA repair systems. This is contrary to group III DNA damage, where a possible relationship between oxidative stress and alteration of nuclear DNA metabolism is found, probably deriving from an antioxidant defence decline.

Detection of MNNG-induced DNA lesions in mammalian cells; validation of comet assay against DNA unwinding technique, alkaline elution of DNA and chromosomal aberrations

Human cells (VH10 or Hep G2) and hamster cells V79 were exposed to different concentrations of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and the level of DNA lesions was evaluated by the DNA unwinding technique, alkaline elution of DNA and the comet assay. All three methods were able to detect the effects of MNNG but with a clear difference in sensitivity. At low concentrations of MNNG the most sensitive method appeared to be the comet assay. After the short-term treatment the comet assay was able to detect the lesions induced by MNNG at approx. 0.1 μg/ml, alkaline elution of DNA at 1 μg/ml and DNA unwinding at 1–2 μg/ml. MNNG treated VH10 cells, human lymphocytes and V79 cells were also tested cytogenetically, confirming that MNNG induced chromosomal aberrations at concentrations >1 μg/ml in VH10 cells (short-term treatment); >0.2 μg/ml in V79 cells (long-term treatment) and >8 μg/ml in human lymphocytes (long-term treatment). In some experiments we tried to increase the level of MNNG-induced DNA breaks with help of DNA repair inhibitors cytosine arabinoside (Ara C) and hydroxyurea (HU) which were applied either after or during MNNG treatment. Our results showed that the level of MNNG-induced lesions was increased by simultaneous treatment of cells with MNNG and Ara C and HU. 2×10−5 M Ara C and 2×10−3 M HU were as effective as 10-times higher concentrations of inhibitors. Ara C and HU increased the level of MNNG-induced DNA breaks mainly in combination with lower concentrations of MNNG (<2 μg/ml). Rejoining of DNA breaks was observed in human cells VH10 and Hep G2 as well as in Chinese hamster cells V79 damaged by both lower and higher MNNG-concentrations. All methods showed that MNNG-induced DNA breaks had been gradually rejoined.

Single‐strand breaks, chromosome aberrations, sister‐chromatid exchanges, and micronuclei in blood lymphocytes of workers exposed to styrene during the production of reinforced plastics

Chromosome aberrations (CA), micronuclei (MN, cytokinesis-block [CB] method), and sister-chromatid exchanges (SCE) were analysed in blood lymphocytes of 17 workers and 17 control subjects. The mean urinary mandelic acid level (average 9.4 mmol/l) and styrene glycol in blood (average 2.5 mumol/l) implied exposure to about 300 mg/m3 of styrene in the plant. The number of CA was significantly higher in non-smoking workers compared with nonsmoking controls. A significant correlation was observed between duration of exposure and individual CA level of all workers. No significant effects were observed in MN or SCE. Single-strand breaks (SSB) in DNA of isolated lymphocytes were studied in nine of the workers and eight of the controls by the DNA-unwinding technique. The results showed an increase in SSB among the exposed workers. The present findings support earlier reports on the increase of structural CA in blood lymphocytes of workers in the reinforced plastic industry, and also show that SSBs are elevated in such workers.

Differences between survival, mutagenicity and DNA replication in MMS- and MNU-treated V79 hamster cells

After treatment with methyl methanesulfonate (MMS) or N-methyl- N-nitrosourea (MNU), the mutagenicity and survival of Chinese hamster V79 cells were investigated, as well as the inhibition of daughter DNA synthesis and, using the DNA unwinding technique and hydroxylapatite chromatography, the character of the newly synthesized DNA was studies. It was found that different cytotoxicity and mutagenicity of MMS and MNU was accompanied by different types of DNA synthesis inhibition. The treatment with the former compund resulted in a longer inhbition of DNA synthesis, while the treatment with the latter showed that as early as 2 h after exposure the percentage of nascent DNA increased. Shortly after the exposure to both alkylating agents, the newly synthesized DNA contained a higher number of gaps than control DNA, in dependence on the concentration used. During culturing after treatment, the character of nascent DNA in MMS-treated cells gradually returned to the of control DNA, while MNU-treated cells, for the whole time of our study, synthesized DNA with a larger number of gaps than control DNA. We suggest that the character of nascent daughter DNA reflects the occurrence og lesoins in parental DNA. These are repaired within a shorter time in MMS than in MNU-treated cells. The long-term persistence of lesions in the DNA of MNU-treated cells might be one of the factors responsible not only for the higher cytotoxic but also for the many times higher mutagenic effect of this alkylating agent.

“The derivative assay”—An analysis of two fast components of DNA rejoining kinetics

The DNA rejoining kinetics of human U-118 MG cells were studied after γ-irradiation with 4 Gy. The analysis of the sealing rate of the induced DNA strand breaks was made with a modification of the DNA unwinding technique. The modification meant that rather than just monitoring the number of existing breaks at each time of analysis, the velocity, at which the rejoining process proceeded, was determined. Two apparent first-order components of single-strand break repair could be identified during the 25 min of analysis. The half-times for the two components were 1.9 and 16 min, respectively.

The tissue specificity of the age-related changes in alkali-induced DNA-unwinding.

To determine if the age-related changes in chromatin digestibility are tissue-specific, fluorometric analysis of the alkali-induced DNA unwinding technique was adapted for soft-tissue chromatin studies. The rate of DNA unwinding in the brain and liver of young Fischer 344 male rats (3 months of age) was significantly greater than the rates measured in middle-aged (15 months) or aged rats (26 months). In contrast, the rate of DNA unwinding in the intestinal epithelium, a continuously replicating tissue, did not significantly vary with age. Although this assay is capable of detecting DNA strand breaks in vivo following N-nitrosodimethylamine administration, the age-related changes could not be attributed to reduced DNA strand lesions in the aged animals. The % double-stranded DNA at time 0 of incubation in alkali was lower in the brain and liver of aged rats indicating that DNA strand breaks may actually increase with aging. These results indicate that proliferative activity of the tissue is an important determinant of age-related changes in chromatin structure.

Differences in inhibition by beta-arabinofuranosyladenine (araA) of radiation induced DNA damage repair in exponentially growing and plateau-phase CHO-cells.

The effect of beta-arabinofuranosyladenine (araA) on the repair of radiation induced DNA damage, as measured by the DNA unwinding technique, was studied in exponentially growing and plateau-phase CHO-cells after exposure to x-rays. Induction of DNA damage by radiation was found to be similar in exponentially growing and plateau-phase cells. In the absence of araA, repair of radiation induced DNA damage proceeded with similar kinetics in exponentially growing and plateau-phase cells. AraA at concentrations between 0-1500 microM inhibited DNA repair both in exponentially growing and in plateau-phase cells. However, the degree of inhibition was significantly higher (by a factor of 3) in plateau-phase cells. A similar degree of repair inhibition by araA was observed in plateau phase cells treated in their conditioned medium, as well as in plateau phase cells that were transfered in fresh growth medium just before treatment initiation. These results indicate the importance of biochemical parameters associated with alterations in the growth state of the cells for the inhibitory effect of araA and may help in the elucidation of the molecular mechanism(s) underlying repair inhibition by inhibitors of DNA replication.

Repair of DNA single-strand and double-strand breaks in the Chinese hamster xrs 5 mutant cell line as determined by DNA unwinding

The DNA unwinding technique has been used to measure the induction and repair of DNA strand breaks by X-rays in the X-ray sensitive (xrs 5) mutant and its parent CHO K1 line of Chinese hamster cells. Results show that frequency of induction of DNA strand breaks was the same for both cell lines. The repair of single-strand breaks was found to be slightly slower in xrs 5 over the first 20 min after X-ray exposure, but the level of repair of ssb reached after an incubation of 1h following X-ray exposure in xrs 5 was the same as in CHO K1. Our results also show that the rate of repair of DNA double-strand breaks in xrs 5 cells was clearly slower than that in CHO K1, supporting the conclusion of Kemp et al. (1984) who used the neutral elution technique, that xrs 5 is defective in the repair pathway of DNA double-strand breaks.

Impaired repair of UVC-induced DNA damage in L5178Y-R cells: DNA unwinding studies with the use of 1-beta-D-arabinofuranosyl cytosine.

Abstract— L5178Y‐R (LY‐R) and L5178Y‐S (LY‐S) cells differ in sensitivity to UVC radiation (D0: 2.8 and 9.0 J/m2respectively, for cells exposed in Fischer's medium). We used these cells and a DNA unwinding technique in conjunction with 1‐β‐D‐arabinosyl cytosine to determine DNA strand breaks accumulating as a result of enzymatic incision during DNA repair. Following UVC exposure DNA strand break accumulation was observed in LY‐S cells but not in LY‐R cells. The repair defect in LY‐R cells is accompanied by a delayed recovery of [3H]thymidine incorporation.

A direct assay for detection of chemically induced changes in the rejoining kinetics of radiations induced DNA strand breaks

A simple and sensitive procedure for testing various chemicals affecting DNA repair is represented. Cells, either labelled with [ 3H]thymidine or [ 14C]thymidine, were drug-treated or used as reference cells. Both cell populations were irradiated with 5 Gy. The number of DNA breaks were determined, after mixing of drug-treated and reference cells of different labelling, at various intervals by the DNA unwinding technique and the drug-dependent DNA breaks were calculated. The drugs benzamide, 3-aminobenzamide,novobiocin and 9-β- d-arabinofuranosyladenine (araA), all known to affect DNA repair, were used to study their effect on the number of DNA strand breaks with the presented technique. It was found that the assay improved the accuracy in determining the influence of DNA repair inhibitors compared to indirect measurements.

Induction of single-strand breaks in dna of mice by trichloroethylene and tetrachloroethylene

Trichloroethylene (TRI) (4–10 mmol kg body wt) and tetrachloroethylene (PER) (4–8 mmol kg body wt) were given to male mice by i.p. injection. The induction of single-strand breaks (SSB) in DNA of liver, kidney and lung was studied by the DNA unwinding technique. There was a linear increase of the level of SSB in kidney and liver DNA but not in lung DNA l h after administration. The damage was completely repaired 24 h after injection. The capability of TRI and PER to induce SSB in liver DNA is compared to that of three other substances, i.e., methyl methanesulfonate (MMS), styrene-7,8-oxide and styrene, which have been studied earlier by the same technique. The potency of the substances for induction of SSB was in the following order: MMS > styrene-7,8-oxide > styrene > PER > TRI.

DNA strand break rejoining in human lymphocytes during the S phase.

Induction and repair of DNA strand breaks in asynchronous and synchronized cultures of human lymphocytes was investigated by using the alkaline DNA-unwinding technique followed by chromatography on hydroxylapatite. Strand break rejoining in exponentially growing human PHA stimulated lymphocytes, irradiated with 20 Gy of X-rays, is temperature-dependent, being fast at 37 degrees C (half-time of a few minutes), and very slow at around 4 degrees C. In synchronized cells irradiated with the same X-ray dose, the repair capacity increases during S phase reaching its maximum when DNA is entirely duplicated.

Single-strand breaks in DNA of various organs of mice induced by styrene and styrene oxide

Styrene and its metabolite styrene oxide were given i.p. to mice. The induction of single-strand breaks (SSB) in DNA was studied with the DNA unwinding technique. The level of SSB in kidney-DNA was a linear function of the dose for both substances. Styrene and styrene oxide induced an increase in the level of SSB in DNA of kidney, liver, lung, testis and brain 1 h after administration. After 24 h the damage remained on an enhanced level in liver, lung and testis after styrene oxide administration and in all organs except liver after styrene administration.