The effect of aging and dietary restriction on DNA repair

DNA repair was studied as a function of age in cells isolated from both the liver and the kidney of male Fischer F344 rats. DNA repair was measured by quantifying unscheduled DNA synthesis induced by UV irradiation. Unscheduled DNA synthesis decreased approximately 50% between the ages of 5 and 30 months in both hepatocytes and kidney cells. The age-related decline in unscheduled DNA synthesis in cells isolated from the liver and kidney was compared in rats fed ad libitum and rats fed a calorie-restricted diet; calorie restriction has been shown to increase the survival of rodents. The level of unscheduled DNA synthesis was significantly higher in hepatocytes and kidney cells isolated from the rats fed the restricted diet. Thus, calorie restriction appears to retard the age-related decline in DNA repair.     

Influence of age,sex and strain on the in vitro induction of unscheduled dna synthesis in rat hepatocyte primary cultures

The activity of chemical-induced unscheduled DNA synthesis was evaluated in hepatocyte primary cultures from Fischer 344 and Sprague-Dawley rats over a period of two years. In this two-year study hepatocytes from both sexes and strains were prepared from animals 2, 8, 14, 20 and 25 months of age and UDS was measured by autoradiography following treatment with N-methyl-AP-vitro-N-nitrosoguanidine and 2-acetylaminofluorine. A dose-related positive response occurred for both compounds throughout the study in hepatocytes from male and female Fischer rats and male Sprague-Dawley rats. The magnitude of the response was greatest in hepatocytes from male Fischer rats and a markedly lower response in unscheduled DNA synthesis occurred in all cultures prepared from animals of both strains and sexes at 20 and 25 months of age. Hepatocytes from female Sprague-Dawley rats showed a low level of unscheduled DNA synthesis with N-methylN-vitro-N-nitrosoguanidine throughout the study. The most striking finding was the absence of a UDS response to 2-acetylaminofuorene by hepatocytes from Sprague-Dawley females at the 8, 14, 20 or 25 month periods. The results indicate an age-related decrease in chemical-induced unscheduled DNA synthesis activity among rats.Abbreviations 2AAF 2-acetylaminofluorine[deDMSO] - dimethylsulfoxide ³H-TdR, meth yl-3H-thymidine - MNNG N-methyl-N-vitro-N-nitrosoguanidine - UDS unscheduled DNA synthesis     

Semiconservative and unscheduled DNA synthesis on mammalian cells and its modification by glyoxylic compounds

The interaction of glyoxal and four other glyoxylic compounds with semiconservative DNA synthesis and with unscheduled DNA synthesis induced by 25-G X-rays on TC-SV40 hamster cells has been studied. Both syntheses were evaluated by measuring the incorporation of [3H-methyl]-thymidine into the newly synthetized DNA. The unscheduled DNA synthesis amounts to 4% of semiconservative synthesis. The modification of both syntheses by the glyoxylic compounds was tested using the products at non-toxic concentrations for the cells. All the glyoxals inhibited semiconservative DNA synthesis and potentiated unscheduled DNA synthesis at rather similar levels. These effects have been compared with the radiosensitizing activity of these glyoxals in the same TC-SV40 cells and no relationship could be established.     

Interindividual variation in the responses of cultured human lymphocytes to exposure from DNA damaging chemical agents: interindividual variation to carcinogen exposure.

Human population variability to standardized doses of N-acetoxy-2-acetylaminofluorene (NA-AAF) and 7, 12-dimethylbenz(a) anthracene (DMBA) was determined in cultured lymphocytes by measuring (a) differential stimulation of unscheduled DNA synthesis after 1 h induction of DNA damage by 10 micrometer NA-AAF, (b) the level of NA-AAF induced chromosome aberrations remaining after 8 h of DNA-repair synthesis, and (c) the level of [3H]DMBA bound to DNA after 18 h incubation of resting lymphocytes in 5 micrometer DMBA. All 3 parameters indicated individual variation to carcinogen exposure and were correlated to the population differences in age, sex, blood pressure and mortality rates. Males always had a greater potential to accumulate DNA-damage than did females regardless of the sampled population. DNA-damage potentials increased with increasing age, blood pressure or mortality rates. There was always proportionally greater DNA-damage potentials in the males than in females. The in vitro response of mature granulocytes to a 10 micrometer NA-AAF dose, as estimated by [3H] thymidine incorporation from unscheduled DNA synthesis, was much lower than lymphocyte response. Nevertheless, individual variations in granulocyte NA-AAF induced unscheduled DNA synthesis paralleled the inter-individual fluctuations observed in the lymphocyte responses to NA-AAF.     

Effects of nicotinamide on NAD and poly (ADP-ribose) metabolism in DNA-damaged human lymphocytes

The effect of nicotinamide on unscheduled DNA synthesis was studied in resting human lymphocytes. In cells treated with UV irradiation or with MNNG, nicotinamide caused a two-fold stimulation of unscheduled DNA synthesis and retarded the rate of NAD⁺ lowering caused by these treatments. Nicotinamide also reduced the burst of poly(ADP-ribose) synthesis caused by MNNG treat-ment. Thus under conditions that it enhances unscheduled DNA synthesis, nicotinamide causes marked effects on the metabolism of NAD⁺ and poly(ADP-ribose). The effect of nicotinamide on unscheduled DNA synthesis was shown to be independent of protein or polyamine synthesis.     

Repair of DNA damaged by methylmethane sulfonate in normal human lymphocytes and in xeroderma pigmentosum

Methylmethanesulphonate has been shown to stimulate an intensive unscheduled DNA synthesis in lymphocytes derived from normal donors as well as in those from patients with xeroderma pigmentosum of the classical form. Somewhat less intensive unscheduled DNA synthesis was observed in cells of a patient suffering from xeroderma pigmentosum. In the case of XPII unscheduled DNA synthesis was greatly reduced which supports the peculiarity of this form of xeroderma pigmentosum.     

Comparison of chromosome aberrations, sister chromatid exchanges and unscheduled DNA synthesis in the evaluation of the mutagenicity of environmental factors

Mutagenic character of formaldehyde in vivo was estimated by determining the level of chromosomal aberrations, sister chromatid exchanges and unscheduled DNA synthesis in human lymphocytes. It was found that in case of occupational exposure to formaldehyde the unscheduled DNA synthesis after thiophosphamide treatment in vitro was inhibited and spontaneous level of chromosomal aberrations increased. A negative correlation observed between the unscheduled DNA synthesis and sister chromatid exchanges indirectly confirmed a connection of these exchanges with the DNA repair. The comparison of the results obtained from evaluation of chromosomal aberrations, sister chromatid exchanges and unscheduled DNA synthesis permits suggesting that these methods estimate different sides of the mutagen interaction with a cell and should be considered as mutually complementary methods but not as interchangeable ones.     

Effects of 2',3'-dideoxythymidine triphosphate on replicative DNA synthesis and unscheduled DNA synthesis in permeable mouse sarcoma cells

2',3'-Dideoxythymidine triphosphate differentially inhibited replicative DNA synthesis in permeable mouse ascites sarcoma cells and unscheduled DNA synthesis in bleomycin-treated permeable cells or in isolated rat liver nuclei. The mode of inhibition of 2',3'-dideoxythymidine triphosphate was competitive with respect to deoxythymidine triphosphate. 2',3'-Dideoxythymidine triphosphate inhibited replicative DNA synthesis with a Ki of 8 microM, whereas unscheduled DNA synthesis was more sensitive, the Ki being 0.5 microM. Referring to the differential sensitivity of DNA polymerases alpha and beta to 2',3'-dideoxythymidine triphosphate and to other related information reported previously, the present results suggested that DNA polymerase alpha is playing a major role in replicative DNA synthesis, and DNA polymerase beta in unscheduled DNA synthesis.     

Inhibition of unscheduled DNA synthesis and repair of potentially lethal X-ray damage by 2-deoxy-D-glucose in yeast.

The effects of the glucose antimetabolite, 2-deoxy-D-glucose (2-DG), on DNA repair (assayed by unscheduled DNA synthesis) and on the repair of potentially-lethal damage (assayed by cell viability after irradiation) have been studied in X-irradiated respiratory-deficient yeast cells (auxotroph for 5'-thymidine-monophosphate). Experimental results show that: (a) both these phenomena can be inhibited by 2-DG; (b) the repair of potentially-lethal damage occurs after the unscheduled DNA synthesis is almost complete; and (c) the repair of potentially-lethal damage can be inhibited by 2-DG even after the completion of the unscheduled DNA synthesis.     

Age-related alterations in plasma membrane glycoprotein content and scheduled or unscheduled DNA synthesis.

WI-38 cells of various ages and SV40-transformed WI-38 cells were examined for differences in plasma membrane composition of glycoproteins and DNA synthesis. Sialic acid per milligram of protein content of the membranes of WI-38 cells decreased with passage of time in culture. Other glycoprotein fractions and alkaline phosphatase activity disappeared in the WI-38 cells with passage of time in culture (Phase III). Studies of DNA repair correlated with changes observed in the plasma membrane glycoprotein content of WI-38 cells over a passage of time in culture were also reported. Both the extent and rate of ultraviolet-induced unscheduled DNA synthesis remained relatively constant during the passage of the WI-38 cells until late phase III. At that time the extent of unscheduled DNA synthesis was measurably reduced. The number of cells in a population of phase III cells able to perform semiconservative DNA synthesis diminished with age in culture but not to an extent capable of explaining the observed changes seen in membrane composition of semiconservative DNA synthesis during passage of the cells in culture. Cells with an extended lifespan SV40-transformed WI-38 (VA 13.2 RA) cells, did not vary in membrane composition, semiconservative DNA synthesis, or unscheduled DNA synthesis over 200 serial subpassages of the cells in culture.     

Inhibition of replicative DNA synthesis and induction of unscheduled DNA synthesis in permeable sarcoma cell by bleomycin.

Unscheduled DNA synthesis was induced by bleomycin in isolated rat liver nuclei and in permeable mouse ascites sarcoma cells. ATP significantly enhanced the bleomycin effect of inducing unscheduled DNA synthesis. Replicative DNA synthesis in permeable mouse ascites sarcoma cells was inhibited by bleomycin. The apparent inhibition or stimulation by bleomycin of in vitro DNA synthesis was thought to be determined by a balance between inhibited DNA replicase activity and induced unscheduled DNA synthesis.     

Use of scintillometric quantitation of unscheduled DNA synthesis in isolated rat hepatocytes for the screening of genotoxic agents

The induction of unscheduled DNA synthesis has been considered as a suitable endpoint for the screening of genotoxic agents. Experimentally, unscheduled DNA synthesis is most frequently measured by autoradiography. The purpose of this report was to examine the usefulness of the liquid scintillation counting technique in measuring unscheduled DNA synthesis response in isolated rat hepatocytes. The various liquid scintillation counting-based unscheduled DNA synthesis assay procedures were examined according to the following groupings: (1) procedures based on the acid precipitation of cellular macromolecules, (2) procedures based on isopycnic gradient centrifugation of solubilized cells, (3) procedures based on nuclei isolation in conjunction with other DNA purification methods, and (4) procedures based on the selective retention of hepatocellular DNA. Limited cases in which test chemicals gave positive unscheduled DNA synthesis response in liquid scintillation counting-based assays and negative unscheduled DNA synthesis response in autoradiography-based assays are presented. It is concluded that liquid scintillation counting-based unscheduled DNA synthesis assays represent an appropriate system for inclusion in carcinogenicity and mutagenicity testing programs.Abbreviations 2-AAF 2-acetylaminofluorene - 2-AF 2-aminofluorene - AFB₁ aflatoxin B₁ - ARG autoradiography - DMN dimethylnitrosamine - LSC liquid scintillation counting - MMS methyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - 4-NQO 4-nitroquinoline-1-oxide - PCA perchloric acid - TCA trichloroacetic acid - UDS unscheduled DNA synthesis     

Unscheduled DNA synthesis: Some statistical thoughts

Statistical interpretation of results of experiments involving unscheduled DNA synthesis is examined from a design standpoint. Most appropriate methods currently in use are evaluated and some modifications and extensions are suggested. Concerns about replication and/or interaction errors are evaluated and methods for their appropriate handling are discussed. It is suggested that methods incorporating both dose-response and heterogeneity statistics should be considered in treating results from unscheduled DNA synthesis experiments. Proper designs for such experiments are emphasized.Abbreviations ANOVA analysis of variance - MSE mean square error - UDS unscheduled DNA synthesis     

DNA damage and repair in gamma-glutamyltranspeptidase-positive and negative hepatocytes in primary culture from carcinogen-treated rats.

Chemically induced DNA fragmentation and unscheduled DNA synthesis were determined in gamma-glutamyltranspeptidase (GGT)-positive and GGT-negative hepatocytes isolated from rat livers subjected to a multistage hepatocarcinogenesis regimen (Solt-Farber), which included 0.05% phenobarbital promotion for 6 weeks (early) or 6 months (late). The results indicated that there was DNA damage in untreated GGT-positive and GGT-negative hepatocytes with either period of promotion compared with normal hepatocytes; however, no statistical difference could be seen between GGT-positive and GGT-negative hepatocytes. DNA damage induced in vitro by the activation-dependent carcinogen dimethylnitrosamine was much less in GGT-positive hepatocytes than in GGT-negative hepatocytes or normal hepatocytes. No significant difference in DNA damage was seen in both GGT-positive and GGT-negative cell populations following treatment with the activation-independent carcinogen ethylnitrosourea (ENU), although DNA damage of GGT-positive hepatocytes was less than that of normal hepatocytes. The background of unscheduled DNA synthesis in both GGT-positive and GGT-negative hepatocytes at either time of promotion was higher than that of normal hepatocytes. The capacity for DNA repair in GGT-positive hepatocytes appeared to be lower than that in GGT-negative hepatocytes. GGT-negative hepatocytes exhibited a lower capacity for DNA repair than that of normal hepatocytes in terms of the rate of unscheduled DNA synthesis elicited by dimethylnitrosamine and ethylnitrosourea in vitro.     

Measurement of the kinetics of DNA repair synthesis after uv irradiation using immunochemical staining of incorporated 5-bromo-2'-deoxyuridine and flow cytometry

The kinetics of unscheduled DNA synthesis in normal human fibroblasts was characterized by flow cytometry utilizing the immunofluorescent detection of 5-bromo-2'-deoxyuridine (BrdUrd) incorporated into cellular DNA during the repair process. Quiescent normal human fibroblasts were irradiated with ultraviolet light and incubated in the presence of BrdUrd during a postirradiation repair period. The amount of unscheduled DNA synthesis was then quantified in the quiescent cells by immunofluorescence staining using monoclonal antibodies against BrdUrd incorporated into the DNA. Significant amounts of unscheduled DNA synthesis were measured after doses as low as 0.1 J/m2 and for time periods as short as 15 min. The initial repair rate was found to be linear with time at all doses tested until repair neared completion. Interestingly, the initial repair rate was constant for doses over the range of 5 to 40 J/m2, whereas the time to completion of repair was dose dependent. These results suggest that above 5 J/m2 in normal human fibroblasts, the repair process is saturated but continues to function until all available regions are repaired. Using this methodology for measuring unscheduled DNA synthesis in combination with second and third flow markers, it is now possible to measure unscheduled DNA synthesis in heterogeneous mixtures of cells.     

In Vivo measurement of unscheduled DNA synthesis and S-phase synthesis as an indicator of hepatocarcinogenesis in rodents

Measurement of chemically induced DNA repair as unscheduled DNA synthesis in rodent liver following in vivo treatment is a useful screen for potential hepatocarcinogens. In addition to measurement of unscheduled DNA synthesis, examination of S-phase synthesis provides an indicator of chemically induced cell proliferation in the liver, which may be a basis for hepatic tumor promotion. Several chemicals and classes of chemicals have been examined using these endpoints. The pyrrolizidine alkaloid riddelline is a potent genotoxic agent in vitro, and in vivo studies confirm this response as riddelline induces significant elevations in unscheduled DNA synthesis and S-phase synthesis in rat liver. Conversely, H. C. Blue dyes #1 and #2 are both potent genotoxic agents in vitro but fail to express this genotoxicity in vivo. H. C Blue #1 induces significant increases in S-phase synthesis in B6C3F1 mouse liver, which correlates with the observed carcinogenicity of this compound. Halogenated hydrocarbons likewise fail to induce unscheduled DNA synthesis in vivo, but many of these compounds do increase hepatic cell proliferation in mice, which may be the principal mechanism of hepatocarcinogenesis in this species.Abbreviations BCMEE bis(2-chloro-l-methylethyl)ether - dThd thymidine - HCB1 H.C. Blue #1 - HCB2 H.C. Blue #2 - UDS unscheduled DNA synthesis     

Weakening of the ultraviolet ray-induced unscheduled DNA synthesis in human lymphocytes in extreme old age

The intensity of unscheduled DNA synthesis was studied in UV-irradiated (10--15 J/m2) peripheral blood lymphocytes of 80--90 years old persons. In these extreme old age persons, reparative DNA synthesis was found sufficiently reduced in comparison with that in middle aged (20--43 years old) ones. The role of DNA repair processes in ageing is under discussion.     

The modifying action of methylmethane sulfonate on unscheduled DNA synthesis in the UV irradiation of human peripheral blood lymphocytes

The value of the unscheduled DNA synthesis after the combined effect of UV radiation and methyl methanesulfonate (MMS) was considerably lower than that upon exposure to UV radiation alone and after two-hour incubation of the culture. These differences were insignificant after 26 h incubation. The result can be attributed to the alkylating effect of MMS on the repair DNA polymerase. With MMS delivered prior to UV irradiation there was an even larger decrease in the unscheduled DNA synthesis with both 2- and 26-hour incubation. The data obtained can be explained by the fact that MMS inhibits an excision endonuclease.     

Unscheduled DNA synthesis in primary cultures of human placentae.

Ultraviolet light-induced unscheduled DNA synthesis in primary cultures of human placentae examined as a function of radiation-dose and repair-incubation period was found to be dependent upon cell type and independent of gestational age. Primary cultures obtained by continuous harvesting of enzymatically released cells from fragments of 11-week and term specimens contained cytotrophoblasts and fibroblasts. Fibroblasts exhibited 3-fold more repair than did cytotrophoblasts from the same organ at both 11 weeks and term.     

Possible involvement of DNA polymerases alpha and beta in bleomycin-induced unscheduled DNA synthesis in permeable HeLa cells

DNA polymerases involved in bleomycin-induced unscheduled DNA synthesis in some permeable human cells and rodent cells were studied by using selective inhibitors (aphidicolin, 2′,3′-dideoxythymidine-5′-triphosphate and N-ethylmaleimide) for DNA polymerases. The results suggest that both DNA polymerases α and β are involved in bleomycin-induced unscheduled DNA synthesis in permeable HeLa-S3 cells and probably in some other permeable human cells (HEp-2, KB and WI-38 VA-13 cells). Bleomycin-induced unscheduled DNA synthesis in some permeable rodent cells ($\text{SR-}\text{C3H}\text{He}$, $\text{Balb}\text{c}$ 3T3, 3Y1 and XC cells) is mostly attributed to DNA polymerase β.