Unwinding Method Research Articles

THE EFFECTIVENESS OF PROGRESSIVE MUSCLE RELAXATION AND DEEP BREATHING TECHNIQUES TOWARD STRESS SCORES OF ADOLESCENTS

Adolescence was characterized by the adolescent identity crisis. Adolescents who experienced difficulty can be able to stress. Adolescents who experience academic stress are caused by exhaustion in learning, so many tasks, and full schedules of activities. Treatment which diminishes was of scholastic pressure thought reformist muscle unwinding and profound breathing unwinding methods. The reason for this exploration was to discover the viability of reformist muscle unwinding and procedures profound breathing unwinding toward stress scores in adolescents in the case of high school students in SMK N 1 Jogonalan, Klaten, Indonesia. During this research a quasi experimental design was used. The respondent for this investigation was the students of class X, each group has 9 participants. A random sampling technique was used for sampling. The instruments used were booklets, leaflets, and DASS. Bivariate factual test utilizing Paired t-test and Independent t-test was used for statistical analysis. The stress score was 19.00 before being given progressive muscle relaxation technique and after given progressive muscle relaxation technique, it was reported 16.33. Further, the stress score was 16.67 before the deep breathing relaxation was given and this was 13.78 after the technique of deep breathing relaxation was given. There was a difference in the viability of reformist muscle unwinding with profound breathing unwinding procedures in a pressure score that appeared p-value > 0.05.

Meat Unwinding Techniques in Kilishi Processing in North Cameroon: Constraints and Innovations

Slicing and low technological quality of meat constitute the major technological constraints of kilishi manufacturing process (slices of meat, dried or smoked, seasoned with a cocktail of ingredients and grilled or not on firewood). The unwinding process remains painful and requires skill coupled with proven dexterity in its production. It was to minimize these constraints that a comparative study of three unwinding modes: along the muscle, the 15cmx5cmx5cm and the successive rolling (innovative method) was conducted to know the best unwinding method on two muscles from a zebu goudali, male, brown dress and aged four years,: Round and thick rib on five steps aging period (rigor-mortis, 24h, 48h, 72h and 96h). The results obtained, showed that meat from the slaughterhouse reached the level of the kilishi units in Maroua-Cameroun after 199±8.4 min in rigor-mortis state and it deserved to be aging at least 24h to improve its firmness and from 48h to facilitate unwinding. The unwinding techniques were significantly different (p≤0.05) and successive rolling appeared as the best unwinding mode which reduced the unwinding time of 500g of meat from 15.61 ± 0.57min to 5.88 ± 0.2 min for round and 14.85 ± 0.65 min at 5.58 ± 0.21 min for thick rib during rigor mortis. These times failed after 96h of aging at 4 ± 2°C at 3.16 ± 0.15 min for the Round at 2.48 ± 0.38 min for Thick rib. Aging alone and / or combined with successive rolling improved the firmness of the meat and significantly reduced the unwinding time. The successive rolling feet for all types of muscles joined, with aging contributed to increase the number of muscles of the carcass that had to be used in the processing of kilishi.

An automated Fpg-based FADU method for the detection of oxidative DNA lesions and screening of antioxidants

The oxidation of guanine to 8-oxo-2′-deoxyguanosine (8-oxo-dG) is one of the most abundant and best studied oxidative DNA lesions and is commonly used as a biomarker for oxidative stress. Over the last decades, various methods for the detection of DNA oxidation products have been established and optimized. However, some of them lack sensitivity or are prone to artifact formation, while others are time-consuming, which hampers their application in screening approaches. In this study, we present a formamidopyrimidine glycosylase (Fpg)-based method to detect oxidative lesions in isolated DNA using a modified protocol of the automated version of the fluorimetric detection of alkaline DNA unwinding (FADU) method, initially developed for the measurement of DNA strand breaks (Moreno-Villanueva et al., 2009. BMC Biotechnol. 9, 39). The FADU-Fpg method was validated using a plasmid DNA model, mimicking mitochondrial DNA, and the results were correlated to 8-oxo-dG levels as measured by LC–MS/MS. The FADU-Fpg method can be applied to analyze the potential of compounds to induce DNA strand breaks and oxidative lesions, as exemplified here by treating plasmid DNA with the peroxynitrite-generating molecule Sin-1. Moreover, this method can be used to screen DNA-protective effects of antioxidant substances, as exemplified here for a small-molecule, i.e., uric acid, and a protein, i.e., manganese superoxide dismutase, both of which displayed a dose-dependent protection against the generation of oxidative DNA lesions. In conclusion, the automated FADU-Fpg method offers a rapid and reliable measurement for the detection of peroxynitrite-mediated DNA damage in a cell-free system, rendering it an ideal method for screening the DNA-protective effects of antioxidant compounds.

Protective effect of deoxynucleotide triphosphates on DNA damage in different mammalian cells exposed to -radiation

DNA is generally considered to be the most critical cellular target when considering the lethal, carcinogenic and mutagenic effects of drugs, radiation and environmental chemicals. So the study aim to the determination the damaging effect of -radiation on DNA and the protective effect of deoxynucleotide triphosphates (dNTPs). The study includes three cell types, lymphocytes, kidney cells of African gree monkey (Vero) and hepatocellular carcinoma of human (HePG2) exposed to 1-5 Gy of -radiation and by using fluorometric analysis of DNA unwinding (FADU) method, DNA damage was measured after radiation. The cells were divided into two groups: The first received 5x10-5 dNTPs from 0-30 minutes after radiation, while the second group was not supplemented with deoxynucleotides. Clonogenic survival for vero and HePG2 cell lines was measured. The results revealed that the increase of irradiation dose precipitates an increase of DNA strand breaks. The slope curve of initial DNA damage and mean inactivation dose (D ) differ between vero and HepG2 cell line by a factor of up 3.5 and 2, respectively. dNTPs have clear ameliorating effect on DNA damage. FADU method can play an important role in the choice of a suitable treatment (radiation or drugs) and its dosage according to measurement of DNA damages in selective malignant tissues. Moreover, using dNTPs mixture can reduce the side effect of these treatment especially after experimentally on live mammals (mice).

UV-B-induced formation of reactive oxygen species and oxidative damage of the cyanobacterium Anabaena sp.: protective effects of ascorbic acid and N-acetyl- l-cysteine

Reactive oxygen species (ROS) are involved in the oxidative damage of the cyanobacterium Anabaena sp. caused by UV-B (280–315 nm) radiation. UV-B-induced overproduction of ROS as well as the oxidative stress was detected in vivo by using the ROS-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Thiobarbituric acid reactive substances (TBARS) and fluorometric analysis of DNA unwinding (FADU) methods were adapted to measure lipid peroxidation and DNA strand breaks in Anabaena sp. Moderate UV-B radiation causes an increase of ROS production, enhanced lipid peroxidation and DNA strand breaks, yielding a significantly decreased survival. In contrast, the supplementation of UV-A in our work only showed a significant increase in total ROS levels and DNA strand breaks while no significant effect on lipid peroxidation, chlorophyll bleaching or survival was observed. The presence of ascorbic acid and N-acetyl- l-cysteine (NAC) reversed the oxidative stress and protected the organisms from chlorophyll bleaching and the damage of photosynthetic apparatus induced by UV-B significantly, resulting in a considerably higher survival rate. Ascorbic acid also exhibited a significant protective effect on lipid peroxidation and DNA strand breaks while NAC did not show a substantial effect. These results suggest that ascorbic acid exhibited significantly higher protective efficiency with respect to DNA strand breaks and survival than NAC while NAC appears to be especially effective in defending the photosynthetic apparatus from oxidative damage.

Oxidative DNA Damage: Biological Significance and Methods of Analysis

All forms of aerobic life are subjected constantly to oxidant pressure from molecular oxygen and also reactive oxygen species (ROS), produced during the biochemical utilization of O2 and prooxidant stimulation of O2 metabolism. ROS are thought to influence the development of human cancer and more than 50 other human diseases. To prevent oxidative DNA damage (protection) or to reverse damage, thereby preventing mutagenesis and cancer (repair), the aerobic cell possesses antioxidant defense systems and DNA repair mechanisms. During the last 20 years, many analytical techniques have been developed to monitor oxidative DNA base damage. High-performance liquid chromatography-electrochemical detection and gas chromatography-mass spectro-metry are the two pioneering contributions to the field. Currently, the arsenal of methods available include the promising high-performance liquid chromatography-tandem mass spectrometry technique, capillary electrophoresis, 32P-postlabeling, fluorescence postlabeling, 3H-postlabeling, antibody-base immunoassays, and assays involving the use of DNA repair glycosylases such as the comet assay, the alkaline elution assay, and the alkaline unwinding method. Recently, the use of liquid chromatography-mass spectrometry has been introduced for the measurement of a number of modified nucleosides in oxidatively damaged DNA. The bulk of available chromatographic methods aimed at measuring individual DNA base lesions require either chemical hydrolysis or enzymatic digestion of oxidized DNA, following extraction from cells or tissues. The effect of experimental conditions (DNA isolation, hydrolysis, and/or derivatization) on the levels of oxidatively modified bases in DNA is enormous and has been studied intensively in the last 10 years.

Comparison of biological effects of DNA damage induced by ionizing radiation and hydrogen peroxide in CHO cells

Purpose: Free OH radicals are considered to be the common mediator of DNA damage after ionizing radiation and oxidative stress. In particular, double-strand breaks (dsb) have a major impact on cell killing after irradiation, while the mechanism of cell killing is less clear for oxidative injury. The latter not only affects DNA, but also equally other cell compartments, such as membranes and mitochondria, which may trigger cell death. This study intended to clarify the relationship between DNA damage induction, repair and cell inactivation for hydrogen peroxide and ionizing radiation. Materials and methods: Chinese hamster ovary (CHO) cells were treated with H 2 O 2 in serum-free medium in combination with/without X-irradiation. DNA damage was measured using the alkaline unwinding method or neutral constant-field gel electrophoresis. Cell survival was recorded using the colony-formation assay. Results: Hydrogen peroxide induced a large number of single-strand breaks (ssb>36000/cell) without impairing cell survival. This number reached a maximum (36Gy-equiv. at 3x10 -4 mol/dm3) without further increase after higher concentrations. Repair kinetics of ssb were similar to those after irradiation. Dsb were found only after very high concentrations of H 2 O 2 (>3x10 -2 mol/dm 3) , which is different from irradiation which generated ssb and dsb in the same dose range. A linear-quadratic increase of dsb was found with increasing concentrations of H 2 O 2 suggesting a single or a pairwise action of OH radicals to form a dsb. After either irradiation or peroxide treatment cell killing was observed only after doses which also allowed dsb detection. The number of dsb calculated per lethal event was in the same range but slightly higher after irradiation (1.7-fold) than after H 2 O 2 treatment. Conclusions: Cell killing after irradiation or hydrogen peroxide appears to be due to dsb, whereas cells withstand large numbers of single-strand lesions and other types of non-DNA damage occurring at lower concentrations of hydrogen peroxide. The number of ssb saturates at intermediate concentrations of H 2 O 2 suggesting that a limited amount of chromatin-bound metal ions is available for OH radical generation.

Cytotoxic and genotoxic effect of inhibitor of vulcanisation N-cyclohexylthiophthalimide in a battery of in vitro assays

Mutagenicity of N-cyclohexylthiophthalimide (Duslin P) was tested first by the Ames test in the bacteria, Salmonella typhimurium. The negative results of the Ames test suggested that this compound does not induce mutations in the genome of S. typhimurium under the conditions used. To estimate the cytotoxicity of Duslin P to human cells, we measured cellular DNA and protein as well as cell proliferation, i.e., the mitotic index of treated and control cells. The genotoxic effects were assayed by two biochemical methods developed for detection of single-strand breaks of DNA in mammalian cells, i.e., by the alkaline single cell gel electrophoresis (comet assay) and by the DNA unwinding method, respectively. The DNA unwinding method showed that this compound did not induce DNA damage at concentrations <7 μg/ml. Alkaline single cell gel electrophoresis revealed approximately double the level of DNA damage (in comparison to untreated control DNA) at a concentration of 2 μg/ml, which reduced proliferation to approximately 30%, and triple the level of DNA damage at higher concentrations (6 and 7 μg/ml), which inhibited completely both DNA synthesis and proteosynthesis. Cells with moderately damaged DNA were more common than cells with heavily damaged DNA. Parallel experiments with the strong mutagen and carcinogen MNNG showed that MNNG induced in cells a high level of DNA damage at concentrations which did not reduce the mitotic index or proteosynthesis, while DNA synthesis inhibited only partially. After treatment with MNNG, cells with heavily damaged DNA were more common than cells with moderately damaged DNA. Duslin P-treated VH10 cells were also tested cytogenetically, confirming that Duslin P induced neither chromosomal aberrations nor aneuploidy. We conclude that Duslin P has no mutagenic effect on bacteria, does not induce chromosomal aberrations and CREST positive or CREST negative micronuclei in human cells and induces only a small increase of DNA damage in human cells which is consistent with DNA fragmentation due to cell death.

Zinc protects against ultraviolet A1-induced DNA damage and apoptosis in cultured human fibroblasts.

Ultraviolet A1 (UVA1) radiation generates reactive oxygen species and the oxidative stress is known as a mediator of DNA damage and of apoptosis. We exposed cultured human cutaneous fibroblasts to UVA1 radiation (wavelengths in the 340-450-nm range with emission peak at 365 nm) and, using the alkaline unwinding method, we showed an immediate significant increase of DNA strand breaks in exposed cells. Apoptosis was determined by detecting cytoplasmic nucleosomes (enzyme-linked immunosorbent assay method) at different time points in fibroblasts exposed to different irradiation doses. In our conditions, UVA1 radiation induced an early (8 h) and a delayed (18 h) apoptosis. Delayed apoptosis increased in a UVA dose-dependent manner. Zinc is an important metal for DNA protection and has been shown to have inhibitory effects on apoptosis. The addition of zinc (6.5 mg/L) as zinc chloride to the culture medium significantly decreased immediate DNA strand breaks in human skin fibroblasts. Moreover, zinc chloride significantly decreased UVA1-induced early and delayed apoptosis. Thus, these data show for the first time in normal cutaneous cultured cells that UVA1 radiation induces apoptosis. This apoptosis is biphasic and appears higher 18 h after the stress. Zinc supplementation can prevent both immediate DNA strand breakage and early and delayed apoptosis, suggesting that this metal could be of interest for skin cell protection against UVA1 irradiation.

UVA-induced oxidative damage to rat liver nuclei: reduction of iron ions and the relationship between lipid peroxidation and DNA damage

Lipid peroxidation and DNA damage and the relationship between the two events were studied in rat liver nuclei irradiated with low dose UVA. Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) by spectrophotometric method and as malondialdehyde-TBA adduct by HPLC, and DNA damage was measured as 8-hydroxy-deoxyguanosine (8-OH-dGu) and strand breakage (or loss of double-stranded DNA) by a fluorometric analysis of alkaline DNA unwinding method. The results show that UVA irradiation by itself increased nuclear lipid peroxidation but caused little or no DNA strand breakage or 8-OH-dGu. When 0.5 mM ferric (Fe +3) or ferrous (Fe +2) ions were added to the nuclei during UVA irradiation, lipid peroxidation and DNA damage, measured both as 8-OH-dGu and loss of double-stranded DNA, were strongly enhanced. Lipid peroxidation occurred concurrently with the appearance of 8-OH-dGu. Fe 3+ ions were reduced to Fe 2+ in this UVA/Fe 2+/nuclei system. Lipid peroxidation and DNA damage were neither inhibited by scavengers of hydroxyl radical and singlet oxygen nor inhibited by superoxide dismutase and catalase. Inclusion of EDTA or chain-breaking antioxidants, butylated hydroxytoluene (BHT) and diphenylamine (an alkoxy radical scavenger), inhibited lipid peroxidation but not the level of 8-OH-dGu. BHT also did not inhibit the loss of double-stranded DNA in this system. This study demonstrates the reduction of exogenous Fe +3 by UVA when added to rat liver nuclei, and, as a result, oxidative damage is strongly enhanced. In addition, the results show that DNA damage is not a result of lipid peroxidation in this UVA/Fe 2+/nuclei system.

Induction of DNA strand breaks in rainbow trout Oncorhynchus mykiss under hypoxic and hyperoxic conditions

Rainbow trout ( Oncorhynchus mykiss) were exposed to various degrees of oxygen saturation (3.3–21.1 mg O 2/l). After 5 h of exposure, trout were killed and DNA single-strand breaks (DSSB) were determind in the gills by the alkaline unwinding method. Significant increases of DSSB compared to normoxic conditions (11 mg O 2/l) were observed under hypoxic and particularly hyperoxic conditions. The highest rate of DSSB occurred when the fish were kept under hypoxic conditions followed by a rapid increase of the oxygen concentration. DSSB formation under these conditions was comparable to that achieved by treatment with 200 μM of the potent carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). These data demonstrate that natural influences, such as varying oxygen concentrations in the water, can be responsible for the induction of DSSB. These findings call into question the usefulness of the measurement of DSSB in fish as an indicator of water pollution by genotoxic chemicals.

Effects of smoking marijuana, tobacco or cocaine alone or in combination on dna damage in human alveolar macrophages

This study examined the role of marijuana smoking in the pathogenesis of human lung cancer by measuring DNA damage in alveolar macrophages (AM). The alkaline unwinding method was used to determine DNA single-strand breaks in AM lavaged from non-smokers [NS] and smokers of marijuana [MS], tobacco [TS] or cocaine [CS], either alone or in combination. DNA damage was related to Superoxide anion (O 2 − ) production by AM stimulated with phorbol myristate acetate (PMA) and to nitric oxide content of smoke using cellular nitrite (NO 2 − ) concentrations. The percentage of double-stranded DNA present after alkaline unwinding was higher in AM of NS (41 ± 5% [11]) and CS (41 ± 4% [9]) versus that of MS (31 ± 4% [8]), TS (35 ± 3% [11]), MTS (26 ± 4% [3]), and CTS ( 27 ± 5% ∗ [10]), mean ± SEM [ n], ∗ = p < 0.1 vs. NS ). PMA stimulated O 2 − production by AM from NS and CS was lower than that of other smokers, but the differences were not significant. O 2 − release, however, had an inverse correlation with DNA single-strand breaks (r = −0.38, p = 0.009). Nitrite content of AM from NS and CS was less than that of other smokers' cells ( p < 0.1 for TS & CTS vs. NS ), but DNA damage had no relationship to NO 2 − concentration. We conclude that AM recovered from MS, either alone or in combination with tobacco smoking, show a trend towards DNA damage. Studies utilizing a larger population should verify our findings and further define its relationship to enhanced oxidant production by macrophages.

Resolution of DNA damage at the single-cell level shows largely different actions of X-rays and bleomycin.

Both X-rays and the radiomimetic agent bleomycin (BLM) induce DNA strand breaks, predominantly via reactive radicals. To compare the induction of breaks with the two agents in Chinese hamster (CHO-K1) cells, two different alkaline unwinding methods, a 3H tracer-based analysis of large cell populations and an optical adaption allowing measurement of single cells, were applied. Radiation and BLM show qualitatively similar dose responses when the average number of DNA strand breaks is measured in a large cell population. However, the breakage pattern at the single-cell level indicates large discrepancies between the actions of the two agents. Irradiated cells show a uniform distribution of DNA strand breaks over the cell population. Effects of treatment with 30 micrograms x ml-1 BLM for 2 hr vary from practically zero in some cells to high levels of DNA strand breakage in others. Unlike the repair of radiation-induced DNA breaks, the repair efficiency of BLM-induced DNA strand breaks, as measured at the single-cell level, varies strongly among cells of the same population. Such heterogeneity at the cellular level potentially reduces BLM's usefulness for tumor therapy because the appearance of BLM-resistant subpopulations may critically impair treatment outcome.

Plant radioresistance and DNA repair efficiency inChlamydomonas reinhardtii andPisum sativum

This paper compares the repair of DNA single strand breaks (ssb) induced by γ-radiation in two strains ofChlamydomonas reinhardtii (137C/+/ and UVS-I) and three lines ofPisum sativum (NN 131, 198, 140) differing in the degree of radioresistance. DNA ssb in cells exposed to γ-rays (50, 100, 200, 500 Gy) were measured by electrophoresis and alkaline unwinding method with subsequent chromatography on hydroxyapatite immediately after irradiation and after 30 min of post-irradiation incubation at 25°C. An increase of double-strand DNA (in%) was found in cells after 30 min post-irradiation incubation.C. reinhardtii strains displayed an equal level of DNA degradation and repair efficiency in the DNA single strand breaks. The radioresistant line N 198 ofP. sativum is characterized by a lower level of induced DNA ssb and higher efficiency of repair of these breaks as compared with less radioresistant lines NN 131 and 140.

Inhibiting the Repair of DNA Damage Induced by Gamma Irradiation in Rat Thymocytes

This study assessed the ability of 11 established and potential radiosensitizing agents to retard the repair of radiation-induced DNA damage with a view to enhancing the immunosuppressive effects of in vivo lymphoid irradiation. The capability of irradiated rat thymocytes to repair DNA damage was assessed by an adaptation of the fluorimetric unwinding method. Three compounds, 3-aminobenzamide (3-AB), novobiocin and flavone-8-acetic acid (FAA), inhibited repair significantly. We also report the effect of low-dose irradiation combined with repair inhibitors on the relationship between DNA strand breaks, fragmentation, cell viability and use of nicotinamide adenine dinucleotide (NAD). DNA fragmentation was increased by 1 mM/1 FAA, 1 mM/l novobiocin and 50 microM/l RS-61443 within 3 h of incubation. The latter two compounds also proved cytotoxic. All three drugs augmented the effect of ionizing radiation on the use of NAD. Of the agents investigated, FAA showed the most promise for augmenting the immunosuppressive action of irradiation at nontoxic, pharmacokinetically achievable concentrations.

Cellular damage by ferric nitrilotriacetate and ferric citrate in V79 cells: interrelationship between lipid peroxidation, DNA strand breaks and sister chromatid exchanges

Ferric nitrilotriacetate (Fe-NTA) and ferric citrate (Fe-citrate) were used to study the cellular damage mediated by iron overload with respect to cytotoxicity, lipid peroxidation, DNA strand breaks and sister chromatid exchanges (SCE). At non-toxic concentrations, Fe-NTA induced lipid peroxidation, DNA strand breaks and SCE in a dose- and time-dependent manner. Comparing the time courses of the different events, the increase in lipid peroxidation seems to be associated with the generation of DNA strand breaks, since both types of cellular damage were observed after 1-3 h of incubation. In contrast, the induction of SCE was low after 24 h and increased after 48 and 72 h treatment, indicating the requirement of other types of DNA damage. Fe-citrate was inactive in the induction of lipid peroxidation and SCE, and no significant number of DNA strand breaks were generated, as determined by the alkaline unwinding method. Our results suggest that the induction of lipid peroxidation and DNA strand breaks by iron overload depend on special features of the iron complex applied, while the chromosomal and genetic effects require site specific DNA damage dependent on intracellular iron metabolism. It is concluded that iron overload is able to induce genetic damage in intact cells provided that iron is present in a bioavailable form.

Application of the DNA alkaline unwinding assay to detect DNA strand breaks in marine bivalves

DNA alkaline unwinding methods were used to detect DNA strand breaks in tissues of marine bivalves following field and laboratory exposures and subsequent recoveries in the laboratory. Field deployments of mussels ( Mytilus edulis) or oysters ( Crassostrea virginica) into two highly contaminated urban estuaries (New Bedford Harbor, Massachusetts, and Elizabeth River, Virginia, respectively) resulted in significantly increased DNA strand breaks in gill tissues. DNA strand breaks did not persist in tissues from oysters that were deployed in the Elizabeth River and then allowed to recover in the laboratory for 7–10 days. Laboratory studies were conducted to characterize the sensitivity and specificity of the DNA unwinding method to detect DNA strand breaks in bivalve tissues. Increased DNA strand breaks resulted from acute exposures to sublethal concentrations of the genotoxic agent N-methyl-N'-nitro-N-nitrosoguanidine, but were not persistent for more than 48 h. Acute or chronic (continuous 7-day) exposures of mussels to sublethal concentrations of copper (not generally considered to be genotoxic) did not result in increased DNA strand breaks. These studies demonstrate that DNA alkaline unwinding methods can be used to reveal potentially genotoxic environmental exposures of bivalves. These methods can be used to evaluate the persistence of DNA damage and species-specific DNA repair capacities.

Improvement of the Alkaline Unwinding Method for the Detection of DNA Damage

The alkaline unwinding method to measure DNA damage was reported by Morris and Sherzer (Environ. Mutagen., 7, 871, 1985). In this study, the various assay conditions of the method were examined to improve the detection sensitivity.The detection sensitivity to hepatic DNA damage induced by N-nitrosodimethylamine (NDMA) increased 2-fold when the assay temperature was increased from 4°C to 10°C or 20°C. Changes of liver homogenate concentration (6-10%) markedly influenced the detection sensitivity when the assay was conducted at 4°C, but not at 10°C or 20°C. In liver from mice administered trichloroethylene, no significant DNA damage was detected by the orignal method. In contrast, significant DNA damage was detected when the unwinding assay was conducted using a 6% liver homogenate at 20°C. The modified condition seems to be suitable for detecting stimulants which induce weak DNA damage.

Viscometry of alkaline cell lysates — The hitherto simplest short-term test for chromatin-interactive agents? Investigations in rat thymic and splenic cells

Alkaline (AL) lysates from thymic cells (T-cells) and splenic cells (S-cells) of the rat were measured by low-shearing glass capillary viscometry. AL-viscometry was compared to the nucleoid sedimentation technique and the alkaline unwinding method. The results obtained in cells treated by (a) DNA strand breaking agents (X-rays, UV-light, doxorubicin, bleomycin, hydrogenperoxide, methylmethanesulfonate (MMS)), (b) intercalating and/or cross-linking substances (ethidium bromide, actinomycin D, mitomycin C, bisbenzimide), (c) the DNA repair inhibitor 3-aminobenzamide (3-AB) and (d) hyperthermia suggest that AL-viscometry may be considered as a very simple, rapid and inexpensive preliminary short-term test for detecting chromatin-interactive agents. Whereas agent- and cell-specific characteristics can be also revealed by AL-viscometry, quantification of specific lesions and conclusions as to the mechanisms of action require additional assays.

The kinetics of DNA damage by bleomycin in mammalian cells

The kinetics of DNA damage by bleomycin (BLM) was assessed by measuring the amount of DNA breakage induced by BLM at different doses, treatment lengths, and treatment temperatures. DNA degradation was measured with the alkaline unwinding method. Comparison of the curves of DNA cleavage by BLM leads to the conclusion that low doses (1–5 μg/ml) and short treatments (5–15 min) produce marked damage in the DNA. High increases in BLM concentration produce relatively small increases in DNA damage above the levels obtained with low doses. Extension of treatment times does not increase the DNA degradation above the rate observed with 15-min treatments. The repair of DNA damage starts at about 15 min after the initiation of treatment. The mending of DNA breaks is very fast and extensive when BLM is no longer present. Repair not only implies the closing of DNA nicks, but very likely the degradation of the BLM molecules intercalated in the DNA interrupting the reactions responsible for the generation of free radicals. Persistence of BLM in the cell environment facilitates the replacement of degraded BLM molecules by new ones. This produces the persistent production of free radicals and the establishment of a balance between the processes of DNA damage and repair.