Split-dose exposure to N-methyl-N'-nitro-N-nitrosoguanidine in BALB/3T3 C1 a31-1-1 cells: evidence of DNA repair by alkaline elution without changes in cell survival, mutation and transformation rates

Dose fractionation of a direct-acting chemical carcinogen, the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), was studied for its concurrent effects on survival, DNA damage and repair, ouabain resistance (Ouar) mutations and neoplastic transformation, in the mouse embryo cell line BALB/3T3 C1A31-1-1. MNNG doses of 0.5, 1 and 2 micrograms/ml were added to the cells either as a single exposure or in two equal fractions separated by 1, 3 or 5 h intervals. No significant difference in cytotoxicity was found when single and split-dose treatments were compared. No recovery from sublethal damage was therefore found in this cell line by split-dose administration of MNNG, although such an effect was found when the same cell line was treated with single and split doses of X-rays. Repair of DNA damage as measured by alkaline elution was studied up to 24 h after a single MNNG exposure (0.5 micrograms/ml). DNA repair was rapid during the first 5 h after treatment and slow thereafter. DNA damage detected after split doses of MNNG at 1 and 5 h intervals was significantly lower than after a corresponding single dose. With both single and split doses, rejoining of single-strand breaks (ssb) was nearly complete after 24 h of repair time. Ouar mutation and neoplastic transformation frequencies were determined for single and split doses of MNNG with the second treatment being given during (1 h) or after (5 h) the period of rapid DNA repair. No significant differences in either effect were detected for dose splitting at any tested dose.     

A technique for the measurement of breakage and repair of DNA alkylated in vivo.

The alkaline elution technique has been adapted for use in the assessment of DNA damage induced in the livers and lungs of mice after administration of an alkylating agent, methylemthanesulfonat (MMS). At 4 h after administration of MMS, damage ot DNA was readily demonstrable; the damage was repaired in liver by 24 h. The lung, particularly of the A/J mouse, exhibited an increased alkaline elution rate when compared to C57BL/6J, and repair was not entirely complete (as judged from the rate of alkaline elution of DNA) by 24 h. The rate of elution was dependent upon temperature. It is believed that this adaptation should have great utility in examining DNA repair in vivo.     

The relationship between DNA repair after alkylation damage and in vitro aging in human T-lymphocytes

In vitro cultures of human peripheral blood lymphocytes, both unstimulated G0 cells as well as phytohemagglutinin (PHA) stimulated cells, have been used in the investigation of DNA repair following different types of damage, including that induced by ultraviolet light, ionizing radiation, and chemical agents. We report here repair of DNA damage in cultured normal human T-lymphocytes after treatment with the alkylating agents, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or methanesulfonate (MMS), as measured by the alkaline elution technique. T-lymphocytes cultured with different sources of T-cell growth factors (TCGFs) were shown to have similar repair levels, as measured by loss of single-strand breaks. However, diminished repair was observed with in vitro culture age when T-cells were cultured with PHA and TCGF for a 3-week period. Cell-cycle analysis performed on asynchronously growing cultures indicated that differential repair with in vitro aging was not cell-cycle-related. Fluorescence Activated Cell Sorting (FACS) was used to determine the percentages of CD4+ and CD8+ T-cell subsets present during the in vitro culture periods. Cultures consisted primarily of CD4+ cells until day 20 when the percentage of CD8+ cells increased to approximately 50% of the T-cell population. Neither the absolute percentages of CD4+ and CD8+ cells not the CD4+/CD8+ ratios correlated with repair rates of cultured T-cells. Therefore, the observed decreases in the repair of alkylating agent-induced damage are not due solely to the change in the CD4+/CD8+ ratio.     

Factors modifying 3-aminobenzamide cytotoxicity in normal and repair-deficient human fibroblasts

3-Aminobenzamide (3-AB), an inhibitor of poly(ADP-ribosylation), is lethal to human fibroblasts with damaged DNA. Its cytotoxicity was determined relative to a number of factors including the types of lesions, the kinetics of repair, and the availability of alternative repair systems. A variety of alkylating agents, UV or gamma irradiation, or antimetabolites were used to create DNA lesions. 3-AB enhanced lethality with monofunctional alkylating agents only. Within this class of compounds, methylmethanesulfonate (MMS) treatments made cells more sensitive to 3-AB than did treatment with methylnitrosourea (MNU) or methylnitronitrosoguanidine (MNNG). 3-AB interfered with a dynamic repair process lasting several days, since human fibroblasts remained sensitive to 3-AB for 36-48 hours following MMS treatment. During this same interval, 3-AB caused these cells to arrest in G2 phase. Alkaline elution analysis also revealed that this slow repair was delayed further by 3-AB. Human mutant cells defective in DNA repair differed in their responses to 3-AB. Among mutants sensitive to monofunctional alkylating agents, ataxia telangiectasia cells were slightly more sensitive to 3-AB than control cells, while Huntington's disease cells had a near-normal response. Among UV-sensitive strains, xeroderma pigmentosum variant (XPV) cells were more sensitive to 3-AB after MMS than were XP complementation group A (A) cells, which responded normally. Greater lethality with 3-AB could be dependent on inability of the mutant cells to repair damage by other processes.     

The human cyclin B1 protein modulates sensitivity of DNA mismatch repair deficient prostate cancer cell lines to alkylating agents

DNA damage caused by alkylating agents results in a G2 checkpoint arrest. DNA mismatch repair (MMR) deficient cells are resistant to killing by alkylating agents and are unable to arrest the cell cycle in G2 phase after alkylation damage. We investigated the response of two MMR-deficient prostate cancer cell lines DU145 and LNCaP to the alkylating agent MNNG. Our studies reveal that DU145 cancer cells are more sensitive to killing by MNNG than LNCaP. Investigation of the underlying reasons for lower resistance revealed that the DU145 cells contain low endogenous levels of cyclin B1. We provide direct evidence that the endogenous level of cyclin B1 modulates the sensitivity of MMR-deficient prostate cancer cells to alkylating agents.     

Decreased DNA repair in familial Alzheimer's disease

Alterations in the capacity of a cell to repair DNA lesions play an important role in a number of human diseases. We and others have demonstrated defective DNA repair of alkylation damage in cells from patients with Alzheimer's disease. It has been hypothesized that this defect is related to the cause of Alzheimer's disease and results in the accumulation of lesions in the central nervous system neurons. One prediction of this hypothesis is that in dominantly inherited Alzheimer's disease, the repair defect will be present in half of the offspring of affected patients long before they develop symptoms of the disease. In order to test the hypothesis that decreased DNA repair is responsible for familial Alzheimer's disease and their at-risk offspring we have studied DNA repair in these individuals after exposure of lymphoblasts to alkylating agents. Our results indicate that cell lines from affected patients repair significantly less damage in 3 h than cell lines from healthy controls. A small number of at-risk individuals were also studied and some of these had lower levels of repair, although more cell lines from individuals in this group must be studied. These findings provide further support for defective DNA repair playing a role in the pathogenesis of Alzheimer's disease.     

Fractionation of DNA from mammalian cells by alkaline elution.

The method of alkaline elution provides a sensitive measure of DNA single-strand length distribution in mamalian cells and is applicable to a variety of problems concerning DNA damage, repair, and replication. The physical basis of the elution process was studied. The kinetics of elution above the alkaline transition pH were found to occur in two phases: an initial phase in which single-strand length is rate limiting, followed by a phase in which elution is accelerated due to the accumulation of alkali-induced strand breaks. The range of DNA single-strand lengths that can be discriminated by elution above the alkaline transition pH was estimated by calibration relative to the effects of x ray, and was found to be 5 X 10(8)-10(10) daltons. Shorter DNA strands elute within the pH transition zone, which extended from pH 11.3 to 11.7 when tetrapropylammonium hydroxide was used as base. This elution was relatively rapid, but was sharply limited by pH, according to the length of the strands: the length of the strands eluted increased with increasing pH. Alkaline elution was inhibited by treatment of cells with low concentrations of nitrogen mustard, a bifunctional alkylating known to cross-link DNA. On investigation of the possibility that DNA subclasses may differ in their elution behavior, satellite L strands were found to elute more slowly from cells exposed to a low dose of x ray than did the bulk DNA.     

Deficient repair of DNA lesions in Alzheimer's disease fibroblasts

DNA strand breaks, resulting from treatment with N-methyl-N'-nitro-N-nitrosoguanidine, were repaired more slowly in four strains of familial Alzheimer's disease fibroblasts than in five strains of fibroblasts from age-matched normals. These results were not due to differences between the two cell types in in vitro ages, in the initial DNA damage or in drug-induced cell lysis. Bleomycin-induced DNA double-strand breaks were repaired equally efficiently by both types of cells. Alzheimer's disease cells may have a DNA repair defect, which may be involved in the pathogenesis of this disease.     

Alkaline elution of rat testicular DNA: detection of DNA cross-links after in vivo treatment with chemical mutagens

The alkaline elution technique was used to measure DNA damage in the rat testis after intraperitoneal injection of 3 chemicals known to cause heritable mutations in rodents. These 3 chemicals are triethylenemelamine (TEM), mitomycin C, and cyclophosphamide. All three of these chemicals produced DNA damage which was readily detectable by alkaline elution. Both TEM and mitomycin C produced DNA interstrand cross-links, although TEM was a more potent cross-linker on an equimolar basis than mitomycin C. Cyclophosphamide produced both DNA cross-links and DNA strand breaks. Alkaline elution in the absence of proteinase K indicated that some of the strand breaks appeared to be closely associated with protein. These studied indicate that the alkaline elution technique is capable of detecting DNA damage in mammalian germ cells produced by chemical mutagens. This technique may prove useful as a screening tool for identifying chemicals which cause heritable mutations in mammals.     

Lack of DNA alterations induced by orotic acid in rat liver as evaluated with two DNA unwinding methods

One percent orotic acid supplemented diet is a promoting treatment in the rat model of liver carcinogenesis. After treatment with this type of diet, DNA alterations were observed using alkaline sucrose gradients and alkaline elution methods. In this work we have utilized two unwinding methods for the detection of DNA fragmentation. One method is a viscosimetric method in which the rate of increase in DNA viscosity with time is related to the rate of alkaline DNA unwinding. The second method measures fluorimetrically the amount of renatured and denatured DNA after different times allowed for alkaline DNA unwinding. These two methods are very sensitive in detecting DNA breaks induced by typical alkylating agents, X-rays and H2O2. The two unwinding methods were clearly negative for the orotic acid supplemented diet. We suggest that the DNA alterations detected with alkaline sucrose gradients and alkaline elution methods, after promoting treatment with orotic acid, are probably different from the DNA breaks induced by typical alkylating agents, X-rays and H2O2.     

Acclimation and repair of DNA damage in recombinant bioluminescent Escherichia coli

AIMS: The aim of this study is to understand different adaptive responses in bacteria caused by three different mutagens, namely, an intercalating agent, an alkylating agent and a hydroxylating agent, and the repair systems according to the type of DNA damage, that is, DNA cross-linking and delayed DNA synthesis, alkylation and hydroxylation of DNA. A recombinant bioluminescent Escherichia coli, DPD2794 with the recA promoter fused to luxCDABE originating from Vibrio fischeri, was used in this study. METHODS AND RESULTS: The recombinant bioluminescent E. coli strain DPD2794, containing a recA promoter fused to luxCDABE from V. fischeri, was used to detect adaptive and repair responses to DNA damage caused by mitomycin C (MMC), and these responses were compared with those when the cells were induced with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and hydrogen peroxide (H2O2). The response ratio between the induced samples and that of the controls decreased suddenly when the induced culture was used in further inductions, indicating a possible adaptive response to DNA damage. DNA damage, or the proteins produced, because of MMC addition does not appear to be completely resolved until the seventh sub-culture after the initial induction, whereas simple damage, such as the base modification caused by MNNG and H2O2, appears to be repaired rapidly as evidenced by the quick recovery of sensitivity. CONCLUSIONS: These results suggest that it takes more time to completely repair DNA damage caused by MMC, as compared with a simple repair such as that required for the damage caused by MNNG and H2O2. Therefore, repair of the damage caused by these three mutagens is controlled by different regulons, even though they all induced the recA promoter. SIGNIFICANCE AND IMPACT OF THE STUDY: Using a bioluminescent E. coli harbouring a recA promoter-lux fusion, it was found that different adaptive responses and repair systems for DNA damage caused by several mutagens exists in E. coli.     

The repair of double-strand DNA breaks correlates with radiosensitivity of L5178Y-S and L5178Y-R cells

To better understand the basis for the difference in radiosensitivity between the variant murine leukemic lymphoblast cell lines L5178Y-R (resistant) and L5178Y-S (sensitive), the production and repair of DNA damage after X irradiation were measured by the DNA alkaline and neutral elution techniques. The initial yield of single-strand DNA breaks and the rates of their repair were found to be the same in both cell lines by the DNA alkaline elution technique. Using the technique of neutral DNA elution, L5178Y-S cells exhibited slightly increased double-strand breakage immediately after irradiation, most significantly at lower doses (i.e., less than 10 Gy). Nevertheless, even at doses that yielded equal initial double-strand breakage of both cell lines, the survival of L5178Y-S cells was significantly less than that of L5178Y-R cells. When the technique of neutral DNA elution was employed to measure the kinetics of DNA double-strand break repair, both cell lines exhibited biphasic fast and slow components of repair. However, the double-strand repair rate was much lower in the radiosensitive L5178Y-S cells than in the L5178Y-R cells (T1/2 of 60 vs 16 min). This difference was more pronounced in the fast-repair component. These results suggest that the repair of double-strand DNA breaks is an important factor determining the radiosensitivity of L5178Y cells.     

Detection of DNA damage and repair by alkaline elution using human lymphocytes

The repair of DNA alkylation damage in human cells is poorly understood. We have adapted the alkaline elution technique for use with human peripheral blood lymphocytes in culture. We have also established conditions necessary for short-term culture of human lymphocytes. Lymphocyte growth which can be maintained for up to 30 days is dependent upon irradiated TK6 feeder cells and T-cell growth factor (crude TCGF). The amount of damage induced by a given concentration of methyl methane-sulfonate (MMS) is dependent upon cell number per ml of growth medium. The DNA damage measured, in lymphocytes, by alkaline elution is a composite of single strand breaks and alkali-labile lesions. Repair of this damage after appropriate recovery periods is also detectable. The irradiated feeder TK6 cells do not contribute to the number of strand breaks detected or the amount of recovery after treatment. This method offers a quick and reproducible means of detecting DNA damage and repair in human T-lymphocytes.     

Chemical oncogenesis in cultured mouse embryo cells in relation to the cell cycle.

Using the C3H/10T 1/2 CL8 line of mouse embryo fibroblasts and three different methods of obtaining cell cycle synchrony, namely arginine or isoleucine deficiency and release from postconfluence inhibition of growth, a sensitive phase for oncogenic transformation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been found. This sensitive phase is located somewhere between the G1/S boundary and a point 4 hr prior to this marker. Methylation of cellular macromolecules by tritiated MNNG is not cycle-dependent in cells synchronized by arginine deficiency. The capacity of cells to repair DNA single strand breaks produced by MNNG was examined by alkaline sucrose sedimentation analysis in cells synchronized by arginine deficiency and treated with MNNG during phases of the cell cycle sensitive and insensitive to oncogenic transformation. Whereas DNA repair was found to be equally rapid in cells treated just before S phase (I), or just after commencement of DNA synthesis (III), transformation was maximal in I. By contrast, cells treated when blocked by arginine deficiency (II) repaired DNA slowly and were not sensitive to malignant transformation. Cells in I and II, which repaired DNA at very different rates, were equally sensitive to MNNG-induced lethality, while cells in III, which repaired DNA at the same rate as cells in I, suffered greater lethality. Thus, in this system it was concluded that there was no direct correlation between DNA repair, as measured by alkaline sucrose sedimentation analysis of prelabeled DNA, and malignant transformation or lethality produced by MNNG. In preliminary experiments malignant transformation induced by cytosine arabinoside (1-beta-D-arabinofuranosylcytosine, ara-C) has been found to occur mainly in S phase, indicating that diverse chemical oncogens may have different sites of action, or that activation of chemical oncogens is cell cycle-specific for some agents.     

Overproduction of the poly(ADP-ribose) polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells.

The zinc-finger DNA-binding domain (DBD) of poly (ADP-ribose) polymerase (PARP, EC 2.4.2.30) specifically recognizes DNA strand breaks induced by various DNA-damaging agents in eukaryotes. This, in turn, triggers the synthesis of polymers of ADP-ribose linked to nuclear proteins during DNA repair. The 46 kDa DBD of human PARP, and several derivatives thereof mutated in its first or second zinc-finger, were overproduced in Escherichia coli, in CV-1 monkey cells or in human fibroblasts to study their DNA-binding properties, the trans-dominant inhibition of resident PARP activity, and the consequences on DNA repair, respectively. A positive correlation was found between the in vitro DNA-binding capacity of the recombinant DBD polypeptides and their inhibitory effect on PARP activity stimulated by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Furthermore, overproduced wild-type DBD blocked unscheduled DNA synthesis induced in living cells by MNNG treatment, but not that induced by UV irradiation. These results define a critical role for the second zinc-finger of PARP for DNA single-stranded break binding and furthermore underscore the importance for PARP to act as a critical regulatory component in the repair of DNA damage induced by alkylating agents.     

Alkaline step elution analysis of gamma-ray induced DNA strand breaks and repair in diploid yeast

Gamma-ray induction of DNA strand breaks and their repair was analysed in the diploid yeast strain D7 (Saccharomyces cerevisiae) by means of the alkaline step elution technique. A dose-dependent increase of DNA strand breakage was observed in the dose range 25-2000 Gy corresponding to 100 and 0.01 per cent survival. When, after exposure to gamma-irradiation, the cells were incubated for 2 h in liquid growth medium, the elution profiles reached the pattern of unirradiated controls, thus indicating the restoration of cellular DNA due to repair. The alkaline step elution analysis is found to be a useful and reproducible technique for studying the induction of DNA strand breaks and repair in yeast. In comparison with other current methods, such as alkaline sucrose gradients and DNA unwinding, this method appears to be more rapid, versatile and easier to handle.     

The repair of bleomycin-induced DNA damage and its relationship to chromosome aberration repair.

Previous studies using the technique of premature chromosome condensation indicated that nearly one-half of the bleomycin-induced chromatid breaks and gaps in CHO cells could be repaired within 1 h (repair starting at 30 min) after treatment. Cycloheximide and streptovitacin A (but not hydroxyurea or hycanthone) inhibited chromosome repair. The purpose of this study was to measure the kinetics of DNA repair after bleomycin treatment using the alkaline elution technique and to determine whether various inhibitors could block this repair. After bleomycin treatment, the major proportion of the repair of DNA damage occurred within 15 min, with significant repair evident by 2 min. This fast repair component was inhibited by 0.2% EDTA. A slower repair component was observed to occur up to 60 min after bleomycin treatment. None of the inhibitors tested were found to have a significant effect on the repair of bleomycin damage at the DNA level. Since chromosome breaks were observed not to begin repair until after 30 min while over 50% of the DNA was repaired by 15 min, these results suggest that the DNA lesions that are repaired quickly are not important in the formation of chromosome aberrations. Further, since cycloheximide and streptovitacin A blocked chromosome repair but had little measurable effect on DNA repair, these results suggest that the DNA lesions responsible for chromosome damage represent only a small proportion of the total DNA lesions produced by bleomycin.     

Differential sensitivity of DNA replication and repair in permeable Escherichia coli exposed to various monofunctional methylating or ethylating agents.

Escherichia coli cells made permeable to deoxynucleoside triphosphates by brief treatment with toluene (permeablized) were used to measure the effect of the following chemical alkylating agents on either DNA replication or DNA repair synthesis: methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-ethyl-N′-nitro-N-nitrosoguanidine (ENNG). Replication of DNA in this pseudo-in vivo system was completely inhibited 10–15 min after exposure to MMS at concentrations of 5 mM or higher or to MNU or MNNG at concentrations of 1 mM or higher. The ethyl derivatives of the alkylating agents were less inhibitory than their corresponding methyl derivatives, and inhibition of DNA replication occurred in the following order: EMS < ENNG < ENU. Maximum inhibition of DNA replication by all of the alkylating agents tested except EMS occurred at a concentration of 20 mM or lower. The extent of replication in cells exposed to EMS continued to decrease with concentrations of EMS up to 100 mM (the highest concentration tested).The experiments in which the inhibition of DNA replication by MMS, MNU, or MNNG was measured were repeated under similar assay conditions except that a density label was included and the DNA was banded in CsCl gradients. The bulk of the newly synthesized DNA from the untreated cells was found to be of the replicative (semi-conservative) type. The amount of replicative DNA decreased with increasing concentration of methylating agent in a manner similar to that observed in the incorporation experiments.Polymerase I (Pol I)-directed DNA repair synthesis induced by X-irradiation of permeablized cells was assayed under conditions that blocked the activity of DNA polymerases II and III. Exposure of cells to MNNG or ENNG at a concentration of 20 mM resulted in reductions in Pol I activity of 40 and 30%, respectively, compared with untreated controls. ENU was slightly inhibitory to Pol I activity, while MMS, EMS, and MNU all caused some enhancement of Pol I activity.These data show that DNA replication in a pseudo-in vivo bacterial system is particularly sensitive to the actions of known chemical mutagens, whereas DNA repair carried out by the Pol I repair enzyme is much less sensitive and in some cases apparently unaffected by such treatment. Possible mechanisms for this differential effect on DNA metabolism and its correlation with current theories of chemically induced mutagenesis and carcinogenesis are discussed.     

Overnight lysis improves the efficiency of detection of DNA damage in the alkaline comet assay

The ability to detect DNA damage using the alkaline comet assay depends on pH, lysis time and temperature during lysis. However, it is not known whether different lysis conditions identify different types of DNA damage or simply measure the same damage with different efficiencies. Results support the latter interpretation for radiation, but not for the alkylating agent MNNG. For X-ray-induced damage, cells showed the same amount of damage, regardless of lysis pH (12.3 compared to >13). However, increasing the duration of lysis at 5 degrees C from 1 h to more than 6 h increased the amount of DNA damage detected by almost twofold. Another twofold increase in apparent damage was observed by conducting lysis at room temperature (22 degrees C) for 6 h, but at the expense of a higher background level of DNA damage. The oxygen enhancement ratio and the rate of rejoining of single-strand breaks after irradiation were similar regardless of pH and lysis time, consistent with more efficient detection of strand breaks rather than detection of damage to the DNA bases. Conversely, after MNNG treatment, DNA damage was dependent on both lysis time and pH. With the higher-pH lysis, there was a reduction in the ratio of oxidative base damage to strand breaks as revealed using treatment with endonuclease III and formamidopyrimidine glycosylase. Therefore, our current results support the hypothesis that the increased sensitivity of longer lysis at higher pH for detecting radiation-induced DNA damage is due primarily to an increase in efficiency for detecting strand breaks, probably by allowing more time for DNA unwinding and diffusion before electrophoresis.