Fluorometric Analysis Of DNA Unwinding Research Articles

P03-01: In vivo genotoxicity assessment in C. elegans via the automated fluorometric analysis of DNA unwinding (FADU) assay

P03-01: In vivo genotoxicity assessment in C. elegans via the automated fluorometric analysis of DNA unwinding (FADU) assay

Differential cytotoxicity induced by the Titanium(IV)Salan complex Tc52 in G2-phase independent of DNA damage.

BackgroundChemotherapy is one of the major treatment modalities for cancer. Metal-based compounds such as derivatives of cisplatin are in the front line of therapy against a subset of cancers, but their use is restricted by severe side-effects and the induction of resistance in treated tumors. Subsequent research focused on development of cytotoxic metal-complexes without cross-resistance to cisplatin and reduced side-effects. This led to the discovery of first-generation titanium(IV)salan complexes, which reached clinical trials but lacked efficacy. New-generation titanium (IV)salan-complexes show promising anti-tumor activity in mice, but their molecular mechanism of cytotoxicity is completely unknown.MethodsFour different human cell lines were analyzed in their responses to a toxic (Tc52) and a structurally highly related but non-toxic (Tc53) titanium(IV)salan complex. Viability assays were used to reveal a suitable treatment range, flow-cytometry analysis was performed to monitor the impact of dosage and treatment time on cell-cycle distribution and cell death. Potential DNA strand break induction and crosslinking was investigated by immunostaining of damage markers as well as automated fluorometric analysis of DNA unwinding. Changes in nuclear morphology were analyzed by DAPI staining. Acidic beta-galactosidase activity together with morphological changes was monitored to detect cellular senescence. Western blotting was used to analyze induction of pro-apoptotic markers such as activated caspase7 and cleavage of PARP1, and general stress kinase p38.ResultsHere we show that the titanium(IV)salan Tc52 is effective in inducing cell death in the lower micromolar range. Surprisingly, Tc52 does not target DNA contrary to expectations deduced from the reported activity of other titanium complexes. Instead, Tc52 application interferes with progression from G2-phase into mitosis and induces apoptotic cell death in tested tumor cells. Contrarily, human fibroblasts undergo senescence in a time and dose-dependent manner. As deduced from fluorescence studies, the potential cellular target seems to be the cytoskeleton.ConclusionsIn summary, we could demonstrate in four different human cell lines that tumor cells were specifically killed without induction of major cytotoxicity in non-tumorigenic cells. Absence of DNA damaging activity and the cell-cycle block in G2 instead of mitosis makes Tc52 an attractive compound for further investigations in cancer treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2538-0) contains supplementary material, which is available to authorized users.

Anthracyclines as radiosensitizers: a Cu(II) complex of a simpler analogue modifies DNA in Chinese Hamster V79 cells under low-dose γ radiation

Hydroxy-9,10-anthraquinones are structural analogues of anthracycline anticancer drugs showing similarity in physicochemical attributes, electrochemical behavior and biophysical interactions. 1,2-dihydroxy-9,10-anthraquinone (Q) and its complexes with Cu(II)/Ni(II) were studied for γ radiation induced modification of DNA in Chinese Hamster V79 cells. The amount of double stranded DNA remaining was ascertained by fluorometric analysis of DNA unwinding using ethidium bromide. Modification of double stranded DNA increased in the presence of Q and Cu(II)-Q when cells were irradiated (0–4.2 Gray). Ni(II)-Q was not that effective. Changing incubation time before recovery of DNA from cells there was evidence for DNA repair that was least for Cu(II)-Q treated cells. Minimum repair in case of Cu(II)-Q treated cells suggest the compound either assists radiation induced damage of agents responsible for repair or interacts with species like H2O2 that assist in repair. Since a hydroxy-9,10-anthraquinone and its Cu(II) complex show radiosensitizing property, anthracyclines that contain a hydroxy-9,10-anthraquinone as the core moiety could also be tried as radiosensitizers in treating cancer. .

Mammalian ribonucleotide reductase subunit p53R2 is required for mitochondrial DNA replication and DNA repair in quiescent cells

In postmitotic mammalian cells, protein p53R2 substitutes for protein R2 as a subunit of ribonucleotide reductase. In human patients with mutations in RRM2B, the gene for p53R2, mitochondrial (mt) DNA synthesis is defective, and skeletal muscle presents severe mtDNA depletion. Skin fibroblasts isolated from a patient with a lethal homozygous missense mutation of p53R2 grow normally in culture with an unchanged complement of mtDNA. During active growth, the four dNTP pools do not differ in size from normal controls, whereas during quiescence, the dCTP and dGTP pools decrease to 50% of the control. We investigate the ability of these mutated fibroblasts to synthesize mtDNA and repair DNA after exposure to UV irradiation. Ethidium bromide depleted both mutant and normal cells of mtDNA. On withdrawal of the drug, mtDNA recovered equally well in cycling mutant and control cells, whereas during quiescence, the mutant fibroblasts remained deficient. Addition of deoxynucleosides to the medium increased intracellular dNTP pools and normalized mtDNA synthesis. Quiescent mutant fibroblasts were also deficient in the repair of UV-induced DNA damage, as indicated by delayed recovery of dsDNA analyzed by fluorometric analysis of DNA unwinding and the more extensive and prolonged phosphorylation of histone H2AX after irradiation. Supplementation by deoxynucleosides improved DNA repair. Our results show that in nontransformed cells only during quiescence, protein p53R2 is required for maintenance of mtDNA and for optimal DNA repair after UV damage.

Ultraviolet-B-induced DNA damage and photorepair in the cyanobacterium Anabaena variabilis PCC 7937

The impact of simulated solar radiation on DNA and the mitigation of DNA-damaging effects by photoreactivation was studied in a cyanobacterium Anabaena variabilis PCC 7937. Cultures were irradiated under 295, 320 and 395 nm cut-off filters as well as seven other filters such as WG 280, WG 295, WG 305, WG 320, WG 335, WG 345 and GG 400. Growth of the test organism was found to be affected mostly under UV-B radiation as compared to PAR and PAR + UV-A radiations. Amplification of 16s rDNA and RAPD profile was significantly affected following exposure of genomic DNA to UV-B radiation. The formation of T<>T CPDs was recorded only in the cultures irradiated with UV-B radiation (i.e., under 295 nm as well as under WG 280, WG 295 and WG 305 nm cut-off filters), but maximum yield was found under 280 nm cut-off filter. Furthermore, the considerable induction of thymine dimers was observed with increasing UV-irradiation times. Fluorometric analysis of DNA unwinding (FADU) assay for UV-induced DNA strand breaks exhibited the maximum loss in the percentage of dsDNA under UV-B radiation followed by UV-A and PAR in comparison to the light control samples. We observed that T<>T CPD repair is light-dependent, since these lesions were more efficiently removed upon exposure to visible light than in the darkness. Blue radiation was found to be the most effective in photoreactivation than any other wavebands of light. Furthermore, the rate of photoreactivation was measured under varying temperatures (10, 20 and 30 °C); the repair rate was found to be the maximum at 20 °C under white fluorescent light. Our results indicate that photoreactivation play an important role in survival of the organism under natural conditions in spite of being exposed to the UV-B component present in the solar drops.

The antioxidant effect of lovastatin and vitamin E on phagocyte-induced DNA damage

1523 Background: Lovastatin is an HMG-CoA reductase inhibitor used in the treatment of hypercholesterolemia. Since chronic inflammation may be important in the process of carcinogenesis, novel anti-inflammatory therapies may be useful in cancer prevention. We investigated the antioxidant properties of lovastatin in the presence and absence of D-L-alpha-tocopherol (vitamin E) as evidenced by their effect on phagocyte-induced genotoxicity in mammalian cells. Methods: MRC5 fetal human lung fibroblasts were obtained at passage 18 and not used beyond passage 24. In some experiments, 106 MRC5 cells were exposed to 106 human phagocytes stimulated with 100nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 30 min. In others, MRC5 cells were incubated with 10 uM lovastatin for 30 min or 10-5M vitamin E for 24 hrs or both prior to the addition of the stimulated phagocytes. After treatment, single-strand breaks were measured by the fluorometric analysis of DNA unwinding. Superoxide anion was measured by following the superoxide dismutase inhibitable reduction of ferricytochrome C at 550 nm. Hydrogen peroxide formation was measured using the method of Pick and Keisari as modified by Frenkel and Chrzan. Results: Lovastatin in its naturally occurring lactone form had no effect on superoxide anion production by stimulated phagocytes. When converted to its acid form, however, superoxide anion formation was inhibited by 36%. Similarly, in its acid form, lovastatin completely inhibited hydrogen peroxide formation by stimulated phagocytes. DNA single-strand breakage was assayed in human lung cells exposed to stimulated phagocytes with and without lovastatin and/or vitamin E. Stimulated phagocytes induced a decrease in double-stranded DNA to 48% of control values which, after pretreatment with lovastatin reverted to 91% of control values. In cells preincubated with lovastatin and vitamin E, double-stranded DNA reverted to 100% of control values. Conclusions: Lovastatin acts as an antioxidant by decreasing oxygen radical production by human phagocytes and may be important in abrogating the carcinogenic effect of chronic inflammation. Since vitamin E protects cell membranes from oxidant stress, the combination with lovastatin has the potential to be effective as an anticarcinogenic strategy. No significant financial relationships to disclose.

Fluorometric Analysis of DNA Unwinding (FADU) as a Method for Detecting Repair-induced DNA Strand Breaks in UV-irradiated Mammalian Cells¶

Fluorometric Analysis of DNA Unwinding (FADU) as a Method for Detecting Repair-induced DNA Strand Breaks in UV-irradiated Mammalian Cells¶

Results from the “Technical Workshop on Genotoxicity Biosensing” on the micro-scale fluorometric assay of deoxyribonucleic acid unwinding

The fluorometric analysis of DNA unwinding (FADU assay) was originally designed for rapid detection of X-ray-induced DNA damage in mammalian cells. This cellular bioassay is based on time-dependent alkaline denaturation of DNA under moderate denaturing conditions (pH 12.2–12.4) starting from ends as well as from all DNA break points (single-strand breaks, SSB; double-strand breaks, DSB; alkali-labile sites, ALS). DNA which remained double-stranded after 30 min of alkaline treatment was detected after neutralisation and immediate fragmentation followed by binding to the Hoechst 33258 dye (bisbenzimide) and fluorescence measures. In the current paper, a modified method was used which allows cell cultivation and chemical treatment in the same microplate (μ-FADU) followed by analysis of 96 samples in a microplate fluorescence reader. Exposure of mammalian cells to chemicals was performed for 60 min on ice thus allowing identification of direct acting substances capable of inducing DNA-strand breaks. As an inter-assay standard the action of hydrogen peroxide was tested in every test plate. The results demonstrate that the μ-FADU assay is suitable to detect the presence of chemically induced strand breaks within 3 h.

DNA damage and glutathione content in radiology technicians

Background: We wanted to investigate the effect of X-rays on mononuclear blood cells (MNCs) and red blood cells (RBCs) of radiology technicians exposed to X-rays in hospital. Methods: DNA damage was detected by fluorometric analysis of DNA unwinding. Glutathione levels were measured with enzymatic method in mononuclear blood cells. Glutathione content and catalase (CAT) activity of erythrocytes, and plasma malondialdehyde (MDA) levels were determined by spectrophometric methods. Results: An insignificant increase in plasma malondialdehyde levels and a significant decrease in mononuclear blood cells glutathione levels were observed in nonsmoking radiology technicians. In smoking radiology technicians, on the other hand, in addition to an increase in plasma malondialdehyde levels, DNA damage was also significantly apparent. Besides mononuclear blood cells' glutathione depletion, the glutathione content of red blood cells was also found to be decreased. Conclusion: It can be suggested that smoking seems to augment the toxic effects of radiation.

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.

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.

OZONE-INDUCED DNA SINGLE STRAND-BREAKS IN GUINEA PIG TRACHEOBRONCHIAL EPITHELIAL CELLS IN VIVO

Two-month-old male guinea pigs (Dunkin-Hartley strain, specific pathogen free), 4 in each group, were exposed to 0.00, 0.45, and 1.00 ppm O 3 for 72 h. The trachea with two main bronchi was removed from each animal after O 3 exposure. The trachea lavage fluid was used to measure the protein content as an index of altered tracheobronchial epithelial (TE) cell membrane permeability after O 3 exposure. The TE cells were isolated and employed for the determination of DNA single-strand breaks (SSBs) by fluorometric analysis of DNA unwinding (FADU). The statistical significance level was set atα = .05. The results show that neither the yield nor the viability of the TE cell from various O 3 treatment groups was different from that of controls. Compared to controls, the protein content was elevated significantly after 0.45 ppm O 3 exposure; however, the amount of DNA SSBs was not. The number of DNA SSBs increased significantly in the 1.00 ppm O 3 exposure group when compared to controls. Regardless of the alkali incubation time at 15°C, the double-stranded DNA left in the alkali TE cell lysate was a linear function of O 3 exposure concentrations.

Adaptation of cyanobacteria to UV-B stress correlated with oxidative stress and oxidative damage.

Cyanobacteria must cope with the negative effects of ultraviolet B (280-315 nm) (UV-B) stress caused by their obligatory light requirement for photosynthesis. The adaptation of the cyanobacterium Anabaena sp. to moderate UV-B radiation has been observed after 2 weeks of irradiation, as indicated by decreased oxidative stress, decreased damage, recovered photosynthetic efficiency and increased survival. Oxidative stress in the form of UV-B-induced production of reactive oxygen species was measured in vivo with the oxidative stress-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate. Photooxidative damage by UV-B radiation, including lipid peroxidation and DNA strand breakage, was determined by a modified method using thiobarbituric acid reactive substances and fluorometric analysis of DNA unwinding. Photosynthetic quantum yield was determined by pulse amplitude-modulated fluorometry. The results suggest that moderate UV-B radiation results in an evident oxidative stress, enhanced lipid peroxidation, increased DNA strand breaks, elevated chlorophyll bleaching as well as decreased photosynthetic efficiency and survival during the initial exposure. However, DNA strand breaks, photosynthetic parameters and chlorophyll bleaching returned to their unirradiated levels after 4-7 days of irradiation. Oxidative stress and lipid peroxidation appeared to respond later because decreases were observed after 7 days of radiation. The survival curve against irradiation time exhibited a close relationship with the changes in photosynthetic quantum yield and DNA damage, with little mortality after 4 days. Growth inhibition by UV-B radiation was observed during the first 7 days of radiation, whereas normal growth resumed even under UV-B stress thereafter. An efficient defense system was assumed to come into play to repair photosynthetic and DNA damage and induce the de novo synthesis of UV-sensitive proteins and lipids, allowing the organisms to adapt to UV-B stress successfully and survive as well as grow. No induction of mycosporine-like amino acids (MAA) was observed during the adaptation of Anabaena sp. to UV-B stress in our work. The adaptation of the cyanobacterium correlated with and could be caused by the oxidative stress and oxidative damage.

Use of rat liver slices for the study of oxidative DNA damage in comparison with isolated rat liver nuclei and HepG2 human hepatoma cells

Tissue slices are a useful biological system for lipid peroxidation studies but their use for DNA damage studies is not well characterized. Hence, the present study investigates DNA damage in rat liver slices, in comparison with isolated rat liver nuclei and HepG2 human hepatoma cells, incubated with ferric nitrilotriacetate (Fe(III)-NTA), bromotrichloromethane (BrCCl 3), bromobenzene (BrB) or 2-nitropropane (2-NP) at 37°C for 2 hr. DNA damage was measured in slices, cells or nuclei after centrifugation as formation of as 8-hydroxy-2′-deoxyguanosine (8-OH-dGu) and loss of double-stranded (dsDNA) due to strand breakage using a fluorometric analysis of DNA unwinding (FADU). Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) released into the medium. The results show that in liver slices and isolated nuclei, Fe/NTA (1 m m/4 m m) induced high levels of TBARS but low levels of 8-OH-dGu, whereas the oxidant induced low levels of TBARS and no formation of 8-OH-dGu in HepG2 cells. In all three systems, inclusion of ascorbate caused dose-dependent formation of 8-OH-dGu, and the levels were similar between liver slices and HepG2 cells but were far higher in isolated nuclei. In liver slices the FADU assay was not applicable due to limited solubilization of DNA from the slice, whereas the assay detected significant loss of dsDNA in HepG2 cells and slight loss in isolated nuclei induced by Fe/NTA with or without ascorbate. Liver slices incubated with 1 mm BrCCl 3, BrB or 2-NP had elevated TBARS but had little or no formation of 8-OH-dGu; none of these oxidants induced lipid peroxidation or DNA damage in HepG2 cells. When liver slices obtained from rats injected with diethylmaleate (to deplete GSH) were incubated with BrCCl 3, BrB or 2-NP, levels of TBARS and 8-OH-dGu increased markedly. Similarly, HepG2 cells with decreased GSH showed marked elevation of TBARS and loss of dsDNA induced by these oxidants, although no formation of 8-OH-dGu was detected. The present study demonstrates the usefulness and limitations of liver slices for DNA damage studies and the importance of cellular GSH in the protection of DNA against environmental toxicants.

Fluorometric analysis of DNA unwinding (FADU) as a method for detecting repair-induced DNA strand breaks in UV-irradiated mammalian cells.

Fluorometric analysis of DNA unwinding (FADU assay) was originally designed to detect X-ray-induced DNA damage in repair-proficient and repair-deficient mammalian cell lines. The method was modified and applied to detect DNA strand breaks in Chinese hamster ovary (CHO) cells exposed to ionizing radiation as well as to UV light. Exposed cells were allowed to repair damaged DNA by incubation for up to 1 h after exposure under standard growth conditions in the presence and in the absence of the DNA synthesis inhibitor aphidicolin. Thereafter, cell lysates were mixed with 0.15 M sodium hydroxide, and DNA unwinding took place at pH 12.1 for 30 min at 20 degrees C. The amount of DNA remaining double-stranded after alkaline reaction was detected by binding to the Hoechst 33258 dye (bisbenzimide) and measuring the fluorescence. After exposure to X-rays DNA strand breaks were observed in all cell lines immediately after exposure with subsequent restitution of high molecular weight DNA during postexposure incubation. In contrast, after UV exposure delayed production of DNA strand break was observed only in cell lines proficient for nucleotide excision repair of DNA photoproducts. Here strand break production was enhanced when the polymerization step was inhibited by adding the repair inhibitor aphidicolin during repair incubation. These results demonstrate that the FADU approach is suitable to distinguish between different DNA lesions (strand breaks versus base alterations) preferentially induced by different environmental radiations (X-rays versus UV) and to distinguish between the different biochemical processes during damage repair (incision versus polymerization and ligation).

Determination of the initial DNA damage and residual DNA damage remaining after 12 hours of repair in eleven cell lines at low doses of irradiation

Purpose : To determine the relationship between DNA damage and radiosensitivity at low doses (1-10Gy) for the initial DNA damage and residual DNA damage remaining after 12-h repair. Materials and methods : Eleven cell lines, normal human lung epithelial L132, HT29 human colon carcinoma, ATs4 human ataxia telangiectasia, normal CHO-K1 hamster, repair-deficient xrs1 and xrs5 mutants, repair-deficient SCID rodent cell line, the human normal fibroblast 1BR.3, human ataxia telangiectasia fibroblast AT1BR and the repair-deficient fibroblasts 180BR.B and 46BR.1 were irradiated with 60 Co γ-rays. Radiosensitivity was measured by clonogenic survival assay. DNA damage was measured by fluorometric analysis of DNA unwinding (FADU). Results : The radiosensitivity in the 11 cell lines ranged from SF2 of 0.02-0.61. By FADU assay, the undamaged DNA at 5-Gy ranged from 56 to 93%. The initial DNA damage and radiosensitivity were highly correlated (r 2 =0.81). After 5-Gy irradiation and 12-h repair, two groups of cell lines emerged. Group 1 restored undamaged DNA to a level ranging from 94 to 98%. Group 2 restored the undamaged DNA to a level ranging from 77 to 82%. No correlation was seen between residual DNA damage remaining after 12-h repair and radiosensitivity. Conclusion : It is shown that the initial DNA damage correlates with radiosensitivity at low doses of irradiation. This suggests that the initial DNA damage must be considered as a determinant for radiosensitivity.

Detection of xanthine oxidase activity products by EPR and HPLC in bronchoalveolar lavage fluid from patients with chronic obstructive pulmonary disease

Xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.1.3.22), a molybdenum-containing hydroxylase that produces superoxide and uric acid from purine substrates and molecular oxygen, is involved in the oxidative stress underlying several human pathologies including lung diseases. An enzymatic activity similar to xanthine oxidase was previously reported in bronchoalveolar lavage fluid of patients with chronic obstructive pulmonary disease (COPD-BAL), by fluorometric analysis of DNA unwinding and cytochrome c reduction kinetics. Here we report the detection of xanthine oxidase activity products by electron paramagnetic resonance (EPR) in presence of the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and reversed-phase high-performance liquid chromatography (RP-HPLC) in COPD-BAL ( n = 14, average age of patients 65 years, range 38–81) and BAL from healthy nonsmoker controls ( n = 6, average age 64 years, range 44–73). Superoxide DMPO adducts were detected in COPD-BAL and in an in vitro system containing xanthine and xanthine oxidase (XA/XO), but not in BAL controls and when superoxide dismutase (SOD, 1000 I.U./ml) was added to COPD-BAL. The HPLC analyses after addition of xanthine showed production of uric acid in COPD-BAL and in the XA/XO system but not in BAL controls. These results support the involvement of xanthine oxidase in the mechanisms of superoxide production by BAL supernatant, which increases oxidative stress in chronic obstructive pulmonary disease.

OZONE-INDUCED DNA STRAND BREAKS IN GUINEA PIG TRACHEOBRONCHIAL EPITHELIAL CELLS

Ozone (O3), the major oxidant of photochemical smog, is thought to be genotoxic and a potential respiratory carcinogen or promoter of carcinogenic processes. Because of oxidative reactions with the mucus in the upper airway, O3 reaction products are able to penetrate into the tracheobronchial epithelial (TE) cells. The carcinogenic effects of O3 on the TE cells are especially of interest since most previous studies have focused on the morphology or permeability changes of tracheas only. Therefore, the objective of this study was to examine the potential O3 genotoxicity in TE cells after an in vivo exposure, using DNA strand breaks as an index. Two-month-old male Dunkin-Hartley guinea pigs, specific pathogen free, 4 in each group, were exposed to 1.0 ppm 03 for 0, 12, 24, 48, 72, or 96 h. Animals exposed to filtered air without 03 exposure were used as controls. After O3 exposure, the trachea with two main bronchi was removed from each animal, and TE cells were isolated and employed for determination of DNA strand breaks by fluorometric analysis of DNA unwinding (FADU). The statistical significance level was set at a=.05. Compared with controls, ozone exposure did not alter the TE cell yield or viability, but caused an increase in protein content in tracheal lavage and an increase in DNA strand breaks. The amount of DNA left in the alkali lysate of TE cells found at 72 h exposure was significantly decreased from controls for 3 different alkali incubation times. An increase of the double-stranded DNA left in the alkali lysate of TE cells was observed at 96 h of exposure and approached the value of 24 h of exposure. The same pattern was seen with all 3 different alkali incubation times at 15°C. One Qd unit was estimated to correspond to 100 strand breaks per cell. The Qd was also used as an indicator for 03 damage. Compared to controls, the Qd increases significantly after 1 ppm 03 exposure for 72 h, regardless of the alkali incubation time at 15°C.

Calcium-dependent DNA damage and adenosine 3',5'-cyclic monophosphate-independent glycogen phosphorylase activation in an in vitro model of acetaminophen-induced liver injury.

Acetaminophen (N-acetyl-p-aminophenol [APAP]) hepatotoxicity is a process characterized by Ca2+ deregulation. Cellular functions utilizing Ca2+ as a second messenger molecule affect both cytosolic and nuclear signal transduction. Many studies have independently shown Ca2+-related effects on target molecules in response to toxic doses of APAP; however, the primary Ca2+ target resulting in liver necrosis has not been determined. We hypothesize that Ca2+-dependent DNA damage is a critical event in liver necrosis caused by alkylating hepatotoxins. In this study, Ca2+-dependent endonuclease activity was determined from DNA single-strand lesions measured by fluorometric analysis of DNA unwinding. The status of cytosolic Ca2+ was determined by measuring Ca2+-dependent activation of glycogen phosphorylase a. Primary cultures of mouse hepatocytes exposed to a toxic concentration of APAP showed twofold and greater increases in glycogen phosphorylase a stimulation at 6 hours, which was reversible with Ca2+-chelating agents. Cell death was preceded by a large decline in intact, double-stranded DNA. Following toxic administration of APAP, the percentage of total double-stranded DNA was significantly reduced by 2 hours. At 6 and 24 hours, genomic integrity was compromised by 26% and 37%, respectively, compared with untreated controls. Hepatotoxic effects of APAP-mediated Ca2+ deregulation were confirmed in both primary mouse hepatocytes and the human hepatoblastoma HepG2 cell line by lactate dehydrogenase (LDH) release and tetrazolium reduction using the 3-4,5-dimethylthiazole-2-yl-2,5-diphenyltetrazolium bromide thiazol blue(MTT) assay. The Ca2+ chelator, ethylene glycol-bis (beta-aminoethyl ether) N',N',N', N'-tetraacetic acid (EGTA), blocked APAP-induced phosphorylase a activation and necrotic cell death, but failed to inhibit phosphorylase a activation by the adenosine 3',5'-cyclic monophosphate (cAMP) analogue, dibutyryl cAMP, indicating little or no contribution of the cAMP pathway to phosphorylase a stimulation during APAP-induced necrotic death. Results with these in vitro models of liver injury are interpreted as supporting the hypothesis that increased Ca2+ availability plays a major role in the progression of APAP-dependent cellular necrosis, and that the nucleus is a critical target for APAP hepatotoxicity.