Measurement of DNA strand breakage and DNA repair induced with hydrogen peroxide using single cell gel electrophoresis, alkaline DNA unwinding and alkaline elution of DNA

Three techniques: single cell gel electrophoresis (SCGE), alkaline elution of DNA (AE), and alkaline DNA unwinding (ADU) were chosen to compare the sensitivity among these methods in detection of DNA damage and repair in human diploid VH10 cell line after short-term exposure to hydrogen peroxide. Using SCGE technique a dose-dependent increase in DNA migration was found in cells exposed to hydrogen peroxide in concentration range from 10 micromol/l to 100 micromol/l. Alkaline DNA unwinding method detected increased level of single strand breaks (ssb) in concentration range from 25 micromol/l to 100 micromol/l of H2O2, and alkaline elution of DNA estimated increased DNA elution rate from concentration 50 micromol/l of H2O2. In a time course study to evaluate the kinetics of DNA repair, both SCGE and ADU techniques showed that the repair of DNA strand breaks is very rapid; the level of ssb in treated cells has returned to near the background level within two hours. After this time damage remaining in the DNA was in the form of oxidised bases as revealed the incubation of treated cells with specific DNA repair endonuclease, formamidopyrimidine-DNA glycosylase.     

Identification of hydroxyhydroquinone in coffee as a generator of reactive oxygen species that break DNA single strands

A component in instant coffee that caused DNA single strand breaks was isolated by successive ethyl acetate:ethanol extraction, silica gel column chromatography and high performance liquid chromatography using a reversed phase column. The active component was identified as hydroxyhydroquinone (HHQ). Incubation of supercoiled pBR 322 DNA with HHQ at 0.1 mM in phosphate buffer (pH 7.4) at 37 degreesC for 1 h caused single strand breaks, and reactive oxygen species, hydrogen peroxide and hydroxyl radical, were involved in DNA breaking by HHQ. Genotoxic effects of HHQ including DNA breaking activity through generation of reactive oxygen species have been well-demonstrated because the component is considered to be an important genotoxic intermediate metabolite of benzene. Occurrence of HHQ in coffee must have an important significance to consider genotoxicity of coffee.     

Differential role of hydrogen peroxide and organic peroxides in augmenting asbestos-mediated DNA damage: implications for asbestos induced carcinogenesis

The incubation of asbestos with DNA in presence of peroxides augmented DNA damage several fold as compared to the damage caused by individual treatments. Asbestos in presence of hydrogen peroxide causes DNA double strand breaks, damage to its deoxyribose sugar moiety and enhanced DNA fidelity. However, only DNA double strand breaks and enhanced DNA fidelity could be recorded in presence of organic hydroperoxide/peroxide but no DNA sugar damage could be observed. Further, the extent of DNA damage could be correlated to the carcinogenic potential of asbestos fibre. Crocidolite, the most carcinogenic variety of asbestos, produces maximum damage to DNA in presence of both hydrogen peroxide and organic hydroperoxide/peroxide while chrysolite which is only a co-carcinogen produces significantly less DNA damage. The observed differences in DNA damage by hydrogen peroxide and organic hydroperoxide/peroxide have been ascribed to the differential reactivity of DNA with hydroxyl and alkoxy/aryloxy free radicals produced respectively from these inorganic and organic peroxides.     

Thyrotropin receptor expression in adrenal, kidney, and thymus

Inflammatory conditions characterized by neutrophil activation are associated with a variety of chronic diseases. Reactive oxygen species are produced by activated neutrophils and produce DNA damage which may lead to tissue damage. Previous studies have shown that activated murine neutrophils induce DNA strand breaks in a target plasmacytoma cell, RIMPC 2394. We studied the effect of a water soluble nitroxide anti-oxidant, Tempol, on murine neutrophil induction of DNA strand breaks in this system. Murine neutrophils were isolated from the peritoneal cavity of BALB/cAn mice after an i.p. injection of pristane oil. Neutrophils were activated by the phorbol ester PMA and co-incubated with RIMPC 2394 cells. Control alkaline elution studies revealed progressive DNA strand breaks in RIMPC cells with time. The addition of Tempol to the incubation mixture prevented DNA damage in a dose dependent fashion. Five mM Tempol provided complete protection. Tempol protection against DNA strand breaks was similar for both stimulated neutrophils and exogenously added hydrogen peroxide. Measurement of hydrogen peroxide produced by stimulated neutrophils demonstrated that Tempol did not decrease hydrogen peroxide concentration. Oxidation of reduced metals, thereby interfering with the production of hydroxyl radical, is the most likely mechanism of nitroxide protection, although superoxide dismutase (SOD) like activity and scavenging of carbon-based free radicals may also account for a portion of the observed protection. The anti-oxidant activity of Tempol inhibited DNA damage by activated neutrophils. The nitroxides as a class of compounds may have a role in the investigation and modification of inflammatory conditions.     

Pneumocystis carinii pneumonia in rheumatoid arthritis patients treated with methotrexate. A report of two cases

OBJECTIVE: The induction and repair of DNA single-strand breaks (SSBs) were measured in primary cultured infant rat lens epithelial cells (RLECs) following hydrogen peroxide (HP) exposure, and the influential factors were detected. METHODS: Utilizing nick translation methodology which utilizes incorporation of labeled nucleotides at the sites of DNA SSBs to sensitively quantitate the damage, we studied the effects of temperature, doses of HP and trace amount of selenium on cell survival. RESULTS: The number of DNA SSBs at 37 degrees was higher than that at 4C (P < 0.01). Significant numbers of SSBs were detected after being insulted by as low as 36.4 mumol/L hydrogen peroxide for 12 minutes 4 degrees (P < 0.05). Repair rapidly initiated and almost completed in 60 minutes after mild damage, and it was unable to repair when the DNA damage was induced by HP at toxic concentration. Trace selenium was added to the culture and incubated for 24 hours, then the number of SSBs was significantly decreased (P < 0.01). CONCLUSIONS: At 37 degrees HP may induce DNA SSBs in RLECs. HP induces the SSBs in a dose dependent manner. The DNA SSBs can not be repaired after severe damage. Trace selenium can decrease the degree of susceptibility to oxidative damage in infant RLECs.     

DNA strand breaks in human nasal respiratory epithelium are induced upon exposure to urban pollution

All organisms have the ability to respond and adapt to a myriad of environmental insults. The human respiratory epithelium, when exposed to oxidant gases in photochemical smog, is at risk of DNA damage and requires efficient cellular adaptative responses to resist the environmentally induced cell damage. Ozone and its reaction products induce in vitro and in vivo DNA single strand breaks (SSBs) in respiratory epithelial cells and alveolar macrophages. To determine if exposure to a polluted atmosphere with ozone as the main criteria pollutant induces SSBs in nasal epithelium, we studied 139 volunteers, including a control population of 19 children and 13 adult males who lived in a low-polluted Pacific port, 69 males and 16 children who were permanent residents of Southwest Metropolitan Mexico City (SWMMC), and 22 young males newly arrived to SWMMC and followed for 12 weeks. Respiratory symptoms, nasal cytology and histopathology, cell viabilities, and single-cell gel electrophoresis were investigated. Atmospheric pollutant data were obtained from a fixed-site monitoring station. SWMMC volunteers spent >7 hr/day outdoors and all had upper respiratory symptoms. A significant difference in the numbers of DNA-damaged nasal cells was observed between control and chronically exposed subjects, both in children (p<0.00001) and in adults (p<0.01). SSBs in newly arrived subjects quickly increased upon arrival to the city, from 39.8 +/- 8.34% in the first week to 67.29 +/- 2.35 by week 2. Thereafter, the number of cells with SSBs remained stable in spite of the continuous increase in cumulative ozone, suggesting a threshold for cumulative DNA nasal damage. Exposure to a polluted urban atmosphere induces SSBs in human nasal respiratory epithelium, and nasal SSBs could serve as a biomarker of ozone exposure. Further, because DNA strand breaks are a threat to cell viability and genome integrity and appear to be a critical lesion responsible for p53 induction, nasal SSBs should be evaluated in ozone-exposed individuals.     

Fanconi anemia group A and D cell lines respond normally to inhibitors of cell cycle regulation

Cells from patients with Fanconi anemia (FA) show decreased viability and decreased chromosome stability after treatment with DNA cross-linking agents, compared to normal cells. FA cells also show a relative accumulation at the G2/M transition after such treatment. This has suggested a possible checkpoint abnormality. In the studies presented here, treatment with hydroxyurea, caffeine or inhibitors of cell cycle kinases did not reveal abnormalities in survival or chromosome stability in FA-A or FA-D cells. Chromosomal breaks introduced by hydrogen peroxide or methyl methanesulfonate accumulated to the same extent in FA-A or FA-D cells as in normal cells. We conclude that FA-A and FA-D cells respond normally to agents known to alter the cell cycle or introduce DNA strand breaks. FA cells process strand breaks and a variety of DNA monoadducts normally. Our results are compatible with repair of DNA crosslinks being slower in FA than in normal cells and FA cells having normal cell cycle checkpoints.     

Protection by inhibition of poly (ADP-ribose) synthetase against oxidant injury in cardiac myoblasts In vitro

Peroxynitrite and hydroxyl radical are reactive oxidants produced during myocardial reperfusion injury. In various cell types, including macrophages and smooth muscle cells, peroxynitrite and hydrogen peroxide cause DNA single strand breakage, which triggers the activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS), resulting in cytotoxicity. Using 3-aminobenzamide and nicotinamide, inhibitors of PARS, we investigated the role of PARS in the pathogenesis of myocardial oxidant injury in H9c2 cardiac myoblasts in vitro. Peroxynitrite (100-1000 microM), hydrogen peroxide (0.3-10 microM) and the NO donor compounds S-nitroso-N-accetyl-DL-penicillamine (SNAP) and diethyltriamine NONOate all caused a dose-dependent reduction of the mitochondrial respiration of the cells, as measured by the mitochondrial-dependent conversion of MTT to formazan. Peroxynitrite and hydrogen peroxide, but not the NO donors caused activation of cellular PARS activity. The suppression of mitochondrial respiration by peroxynitrite and hydrogen peroxide, but not by the NO donors, was ameliorated by pharmacological inhibition of PARS. The protection by the PARS inhibitors diminished at extremely high concentrations of the oxidants. Hypoxia (1 h) followed by reoxygenation (1-24 h) also resulted in a significant activation of PARS, and caused a suppression of mitochondrial respiration, which was prevented by inhibition of PARS. Similar to the results obtained with the pharmacological inhibitors of PARS, a fibroblast cell line which derives from the PARS knockout mouse was protected against the suppression of mitochondrial respiration in response to peroxynitrite and reoxygenation, but not to NO donors, when compared to the result of cells derived from wild-type animals. Based on our data, we suggest that activation of PARS plays a role in the myocardial oxidant injury.     

A mechanistic analysis of nitric oxide-induced cellular toxicity

Nitric oxide (NO.)-induced toxicity was investigated in two different cell lines, Chinese hamster ovary (CHO-AA8) and human lymphoblastoid (TK6), over a range of NO. doses (0-9 mM) delivered for an exposure of 2 h. To determine both short-term and delayed effects leading to death, a range of assays was employed to decipher the major mechanisms of cytotoxicity. Examples of damage parameters measured in this study include inhibition of DNA synthesis, damage to mitochondria, loss of cell membrane integrity, apoptosis, changes in cell cycle distribution, and the occurrence of DNA strand breaks. Our results indicate that NO.-induced toxicity is an extremely complex process involving multiple pathways generally leading to apoptotic cell death. Results consistently demonstrate that TK6 cells are much more susceptible to NO.-induced toxicity than CHO-AA8 cells. This difference in sensitivity could be seen for all types of cellular damage examined. The earliest observable effect of NO. exposure is inhibition of DNA synthesis which is not the result of inhibition of ribonucleotide reductase but may be the result of DNA damage leading ultimately to cell cycle arrest.     

Opposite effects of nitric oxide donors on DNA single strand breakage and cytotoxicity caused by tert-butylhydroperoxide

1. The effects of three different NO donors on tert-butylhydroperoxide (tB-OOH)-induced DNA cleavage and toxicity were investigated in U937 cells. 2. Treatment with S-nitroso-N-acetyl-penicillamine (SNAP, 1-30 microM), while not in itself DNA-damaging, potentiated the DNA strand scission induced by 200 microM tB-OOH in a concentration-dependent fashion. The enhancing effects of SNAP were observed with two different techniques for the assessment of DNA damage. Decomposed SNAP was inactive. S-nitrosoglutathione (GSNO, 300 microM) and (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (DETA-NO, 1 mM) also increased DNA cleavage generated by tB-OOH and these responses, as well as that mediated by SNAP, were prevented by the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazolin-1-oxyl-3-oxide (PTIO). 3. SNAP neither inhibited catalase activity nor increased the formation of DNA lesions in cells exposed to H2O2. Furthermore, SNAP did not affect the rate of rejoining of the DNA single strand breaks generated by tB-OOH. 4. Under the conditions utilized in the DNA damage experiments, treatment with tB-OOH alone or associated with SNAP did not cause cell death. However, SNAP as well as GSNO markedly reduced the lethal response promoted by millimolar concentrations of tB-OOH and these effects were abolished by PTIO. Decomposed SNAP was inactive. 5. It is concluded that low levels of NO donors, which probably release physiological concentrations of NO, enhance the accumulation of DNA single strand breaks in U937 cells exposed to tB-OOH. This NO-mediated effect appears to (a) not depend on inhibition of either DNA repair (which would increase the net accumulation of DNA lesions by preventing DNA single strand break removal) or catalase activity (which would also enhance the net accumulation of DNA lesions since H2O2 is one of the species mediating the tB-OOH-induced DNA cleavage) and (b) be caused by enforced formation of tB-OOH-derived DNA-damaging species. In contrast to these results, similar concentrations of NO prevented cell death caused by millimolar concentrations of tB-OOH. Hence, DNA single strand breakage generated by tB-OOH in the absence or presence of NO does not represent a lethal event.     

Reading level, learning presentation preference, and desire for information among cancer patients

Protease inhibitors such as aprotinin and urinastatin inhibited vascular endothelial cell injury induced by PMA-stimulated leukocytes, although their inhibitors did not suppress the production of active oxygen species released from leukocytes. On the other hand, in the presence of pancreas elastase (10 micrograms/ml), hydrogen peroxide (50 microM) caused severe injury of endothelial cells isolated from the bovine carotid artery (% specific 51Cr release, % SR = 42.9 +/- 3.3%), although the % SR elicited by elastase or hydrogen peroxide alone, respectively, was below 1%. Elastase and hydrogen peroxide acted synergistically on the injury of endothelial cells from the bovine carotid artery similarly to that in the endothelial cells isolated from the bovine coronary artery and human umbilical vein. Furthermore, elastase derived from both pancreas and leukocyte induced this synergistic action on endothelial cell injury. To clarify the mechanism of vascular endothelial cell injury induced by the combination of elastase and hydrogen peroxide, we examined the effects of various radical scavengers and protease inhibitors. Deferoxamine mesylate completely inhibited the endothelial cell injury, while protease inhibitors such as antitrypsin and macroglobulin had a protective effect. Pretreatment of endothelial cells with deferoxamine mesylate also protected against this cytotoxicity. These findings suggested that the synergistic effect of elastase and hydrogen peroxide on the endothelial cell injury is due to the production of hydroxylradical in the endothelium and that this synergistic action might be partially involved in the endothelial cell injury induced by activated leukocytes.     

Asbestos and DNA double strand breaks

A radiosensitive DNA repair-deficient xrs-5 cell line was used to study asbestos cytotoxicity and DNA double strand breaks (DSBs). Although xrs-5 cells did not show any increase in sensitivity to chrysotile fibers in short-term (4-h) treatment when compared with wild-type CHO cells, longer-term exposure (24 h) gave significantly lower cell survival accompanied by a cell growth delay as well as a higher DNA DSB induction in this mutant cell line. These results suggest an important role played by DNA DSBs at the initial stage of asbestos injury.     

Protective effect of ebselen against hydrogen peroxide-induced cytotoxicity and DNA damage in HepG2 cells

The protective effect of ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a selenoorganic compound, against hydrogen peroxide (H2O2)-induced cytotoxicity and DNA damage was investigated in a human hepatoma cell line, HepG2. The inhibitory effect of H2O2 on cell growth was determined using the tetrazolium dye colorimetric test (MTT test), and the cytotoxicity and lipid peroxidation were estimated by lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) formation, respectively. DNA damage was detected using single cell gel electrophoresis (comet assay), and intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA). The results showed that H2O2 suppressed the growth of HepG2 cells and the addition of ebselen significantly reduced the suppression. Furthermore, ebselen also displayed a dose-dependent reduction of LDH leakage and MDA formation in H2O2-treated cells. The results also demonstrate that ebselen was able to reduce the ROS formation and DNA damaging effect caused by H2O2 in a dose-dependent manner. These findings suggest that ebselen has a strong protective ability against the cytotoxicity and DNA damaging effect caused by reactive oxygen species.     

Self and object in countertransference

We investigated the cloning efficiency, DNA repair, and the rate of DNA replication in the skin fibroblasts from patients with Werner's syndrome (WS) of an autosomal recessive premature aging disease. Five WS strains exhibited normal levels of sensitivity toward X-ray and UV killings and repair of X-ray induced single strand breaks of DNA (rejoining) and UV damage to DNA (unscheduled DNA synthesis). The sedimentation of newly synthesizing DNA in alkaline sucrose gradients demonstrated a characteristic feature that only the elongation rate of DNA chains, estimated by the molecular weight increase, was significantly slower during early passages in WS cells than in normal Hayflick Phase II fibroblasts. In addition, plating efficiencies as well as the replicative potentials of five WS strains were more limited than those of normal cells under the identical culture conditions. It seems therefore that at least in the WS cells tested, the slow rate of DNA replication may be more related to the shortened lifespan and enhanced cell death, as manifestation of premature senescence at the cellular level, than be the DNA repair ability.     

Hydrogen peroxide causes RAD9-dependent cell cycle arrest in G2 in Saccharomyces cerevisiae whereas menadione causes G1 arrest independent of RAD9 function

This study shows differences at the level of cell cycle arrest between the response of yeast cells to hydrogen peroxide and superoxide stress. These include both cell cycle phases at which arrest occurs and the involvement of the RAD9 checkpoint gene. Wild-type and rad9 cells were treated with hydrogen peroxide or the superoxide-generating agent menadione. rad9 mutants were up to 100-fold more sensitive to hydrogen peroxide but not affected in their resistance to menadione. Hydrogen peroxide caused G2-phase arrest, whereas menadione-treated cells arrested in G1. G2 arrest, induced by methyl 2-benzimidazil carbamate, increased cellular resistance to hydrogen peroxide but not to menadione. G1 arrest mediated by alpha-factor caused an increase in survival of wild-type cells treated with menadione but not with hydrogen peroxide. A cdc28 mutant arrested in G1 was significantly more sensitive to hydrogen peroxide than other cdc mutants arrested in later phases, including G2. rad9 cells have normal stationary phase resistance to hydrogen peroxide, the ability to adapt to it, glutathione content and induction of genes via the stress responsive element. Although rad9-dependent G2 arrest is important, other rad9-dependent factors may be involved in the resistance of cells to hydrogen peroxide since arrest in G2 did not make rad9 cells fully resistant.     

Airway inflammatory effect of hydrogen peroxide in guinea pigs

Reactive oxygens are now considered to be important substances in promoting inflammatory process. Recently, airway inflammation has attracted attention closely linked to bronchial asthma. The present study was undertaken to examine whether hydrogen peroxide, one of the reactive oxygens, could produce airway inflammation. Airway inflammation was assessed by airway vascular permeability in terms of pontamine sky blue (PSB) exudation. Airway resistance was measured with a modified Konzett-R?ssler method and was expressed as a change in ventilation overflow. Inhalation of hydrogen peroxide (0.01-1.0 M) markedly caused a PSB exudation in a concentration-dependent manner in all of the trachea, main bronchus, and lungs. The hydrogen peroxide-induced PSB exudation effect was attenuated was attenuated by pretreatment with catalase, although heat-inactivated catalase had no inhibitory effect. Deferoxamine, which inhibits conversion of hydrogen peroxide into hydroxyl radical, decreased the PSB exudation induced by hydrogen peroxide. On the other hand, inhalation of hydrogen peroxide (1.0 M) caused a significant and biphasic increase in ventilation overflow. This airway constriction was suppressed by pretreatment with inhaled catalase, but not by inhaled deferoxamine. These results indicate that hydrogen peroxide causes an intense airway inflammation; this inflammatory effect may be mediated not only by hydrogen peroxide itself but also by hydroxyl radical. Hydrogen peroxide and hydroxyl radical may thus play an important role in bronchial asthma and bronchitis through inducing airway inflammation.     

Detection of DNA damage induced by human carcinogens in acellular assays: potential application for determining genotoxic mechanisms

Positive outcomes of in vitro genotoxicity tests may not always occur as a consequence of direct reaction of a compound or a metabolite with DNA. To follow-up positive responses in in vitro tests, we developed two supplemental, cell-free assays to examine the potential of compounds and metabolites to directly damage DNA. Calf thymus DNA was used as the target for the direct detection of adducts by 32P-postlabeling/TLC and electrochemical detection, and alkaline gel electrophoresis was used to detect single-strand breakage of bacteriophage lambda DNA. To show that these assays would detect damage from relevant compounds, we examined nine human carcinogens (aflatoxin B1, busulfan, chlorambucil, cyclophosphamide, diethylstilbestrol, melphalan, 2-naphthylamine, phenacetin and potassium chromate). Each of the nine compounds produced a positive result for one or both endpoints. Using multifraction contact-transfer TLC, we detected 32P-labeled DNA adducts produced by aflatoxin B1, chlorambucil, diethylstilbestrol, melphalan, 2-naphthylamine, and potassium chromate (plus hydrogen peroxide). Aflatoxin B1, diethylstilbestrol and 2-naphthylamine required metabolic activation (induced rat liver S9) to generate DNA adducts. Although potassium chromate alone induced a slight increase in the content of 8-hydroxydeoxyguanosine (a promutagenic adduct produced by reactive oxygen species), addition of hydrogen peroxide greatly increased 8-hydroxydeoxyguanosine levels. The damage to lambda DNA by each human carcinogen (or metabolites), except diethylstilbestrol, was sufficient to generate single-strand breaks after neutral thermal hydrolysis at 70 degrees C. Chromate was a weak inducer of DNA fragmentation, but adding hydrogen peroxide to the reaction mixtures dramatically increased the DNA strand breakage. Our data suggest that these non-routine, acellular tests for determining direct DNA damage may provide valuable mechanistic insight for positive responses in cell-based genetic toxicology tests.     

DNA damaging effects of pesticides measured by the single cell gel electrophoresis assay (comet assay) and the chromosomal aberration test, in CHOK1 cells

One herbicide (isoproturon), two fungicides (carbendazim and chlorothalonil) and etoposide (an effective antitumor agent used as a positive control), were tested for their ability to induce cytotoxic and genotoxic effects in Chinese Hamster Ovary (CHOK1) cells. Etoposide induced DNA damage detectable both by the alkaline Single Cell Gel Electrophoresis (SCGE) assay and the chromosomal aberration (CA) test in absence of noticeable cytotoxicity. With the SCGE assay, a clear induction of DNA damage was observed for chlorothalonil within a 0.2 to 1 microM concentration range. In the CA test, chlorothalonil gave also positive results, inducing mainly chromosome breaks. In contrast, no DNA damage was observed with the SCGE assay for carbendazim and isoproturon. In the CA test, carbendazim induced only numerical aberrations in the concentration range of 25 microM to 100 microM, and isoproturon did not induce any significant increase in CA. In conclusion, chlorothalonil appears genotoxic in proliferative CHOK1 cells, and as expected, the aneugenic compound, carbendazim, did not induce DNA strand breaks in the SCGE assay.     

Clustered organization of S100 genes in human and mouse

Hydrogen peroxide (H2O2) has been reported to be present at significant levels in the lens and aqueous humor in some cataract patients and suggested as a possible source of chronically inflicted damage to lens epithelial (LE) cells. We measured H2O2 effects on bovine and mouse LE cells and determined whether LE cells from old calorically restricted mice were more resistant to H2O2-induced cellular damage than those of same age ad libitum fed (AL) mice. Bovine lens epithelial cells were exposed to H2O2 at 40 or 400 microM for 2 h and then allowed to recover from the stress. The cells were assayed for DNA damage, DNA synthesis, cell viability, cell morphology, response to growth stimuli, and proliferation potential. Hydrogen peroxide-treated cells showed an increased DNA unwinding 50% greater than that for untreated controls. These DNA strand breaks appeared to be almost completely rejoined by 30 min following removal of the cells from a 2-h exposure. The 40 microM exposure did not produce a significantly lower DNA synthesis rate than the control, it responded to growth factor stimuli, and it replicated as did the control cells after removal of H2O2. The 400 microM H2O2 severely affected DNA synthesis and replication, as shown by increased cell size and by markedly reduced clonal cell growth. The cells did not respond to growth stimulation by serum or growth factors and lost irreversibly the capacity to proliferate. The responses of LE cells from old adlib diet (AL) and calorically restricted (CR) mice to H2O2 were significantly different. Exposure of LE cells to 20, 40, or 100 microM H2O2 for 1 h induces a significant loss of cellular proliferation in cells from old AL mice. LE cells from long-term CR mice of the same strain and age were more resistant to oxidative damage at all three concentrations of H2O2 than those of both old and young AL mice and showed a significantly higher proliferation potential following treatment. It is concluded that CR results in superior resistance to reactive oxygen radicals in the lens epithelium.     

Conditional expression of wild-type topoisomerase II complements a mutant enzyme in mammalian cells

Alterations in the amino acid composition, phosphorylation pattern, or intracellular levels of topoisomerase II have been associated with resistance to antineoplastic agents whose effects are mediated through interactions with this enzyme. To develop a model system with which to investigate the determinants of topoisomerase II sensitivity or resistance to antineoplastic agents that target this enzyme, a cDNA encoding the wild-type Drosophila melanogaster topoisomerase II was ligated into a mammalian expression vector containing a glucocorticoid-inducible mouse mammary tumor virus promoter and transfected into an epipodophyllotoxin-resistant Chinese hamster ovary cell line (VPM(r)-5). In two transfectants carrying an intact, full-length Drosophila topoisomerase II cDNA, exposure to the inducing agent, dexamethasone (10 microM), resulted in complementation of the endogenous mutant topoisomerase II and phenotypic reversion to etoposide sensitivity. In the presence of glucocorticoid, etoposide-induced cytotoxicity increased 20-fold, despite the fact that Drosophila topoisomerase II mRNA expression was only 0.1% of that of the endogenous mammalian topoisomerase II. Induced cells demonstrated a marked increase in DNA single strand breaks compared with uninduced resistant cells, thereby providing biochemical evidence supporting increased DNA strand cleavage due to activation of the Drosophila enzyme. These observations demonstrate the ability of a wild-type Drosophila topoisomerase II to complement a mutant mammalian enzyme and suggest that transfectants capable of conditional topoisomerase II expression represent a useful model for studies of the biochemical pharmacology and structure-function relationships of normal and mutant enzymes.