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     

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     

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.     

Kinetics of unscheduled DNA synthesis in UV-irradiated chicken embryo fibroblasts

Unscheduled DNA synthesis induced by 254-nm UV radiation in chicken embryo fibroblasts was examined for 24 h following irradiation, while cells were kept in the dark. The effect on this repair process of a 2-4 h exposure to photoreactivating light immediately after UV was studied. Initial [3H]thymidine incorporation in the light-treated cells was only slightly different from that in cells not exposed to light, but a distinct difference in rate and cumulative amount of unscheduled DNA synthesis was seen several hours after irradiation. By varying the UV dose and the time allowed for photoreactivation, the amount of dimers (determined as sites sensitive to a M. luteus UV-endonuclease) and non-dimers could be changed. The results of these experiments suggest that excision repair of dimers, rather than non-dimer products, is responsible for the unscheduled DNA synthesis seen after UV irradiation.     

The early effects of chemical carcinogens on adult rat hepatocytes in primary culture. II. Effects on unscheduled DNA synthesis, cell division and alpha-fetoprotein production.

The effects of exposure of rat hepatocytes in primary maintenance culture to chemical carcinogens has been studied with respect cytotoxicity and alterations in mitotic index, unscheduled DNA synthesis and alpha-fetoprotein (AFP) production. All compounds tested produced cytotoxicity. Increases in mitotic index and unscheduled DNA synthesis and the production of AFP were observed after treatment of the cells with the carcinogens but not after treatment with the non-carcinogenic isomers. These increases were dose-dependent and depended on the time of exposure and the time incubated postexposure. The patterns of the increase in mitotic index and AFP production after cessation of carcinogen exposure were very similar, with the increase in mitotic index occurring slightly before that for the AFP production and it is suggested from this and other data that the production of AFP is dependent on the generation of a cell species functionally distinct from the non-dividing hepatocytes. It is also suggested that measurement of unscheduled DNA synthesis in conjunction with that of AFP production in cultured hepatocytes may be useful as part of a screening programme for chemical carcinogens.     

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.     

Age-related changes in unscheduled DNA synthesis by rat hepatocytes

Ultraviolet-induced unscheduled DNA synthesis was studied as a function of age in hepatocytes isolated from 6- to 32-months-old rats. Unscheduled DNA synthesis was measured by both DNA specific activity and autoradiography. Using both procedures, a significant decline in unscheduled DNA synthesis was observed after 14 months of age.     

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     

Unscheduled DNA synthesis tests. A report of the U.S. Environmental Protection Agency Gene-Tox Program

The utility of unscheduled DNA synthesis (UDS) testing for screening potentially hazardous chemicals was evaluated using the published papers and technical reports available to the UDS Work Group. A total of 244 documents were reviewed. Based on criteria defined in advance for evaluation of the results, 169 were rejected. From the 75 documents accepted, results were reviewed for 136 chemicals tested using autoradiographic approaches and for 147 chemicals tested using liquid scintillation counting (LSC) procedures; 38 chemicals were tested by both approaches to measure UDS. Since there were no documents available that provided detailed recommendations of UDS screening protocols or criteria for evaluating the results, the UDS Work Group presents suggested protocols and evaluation criteria suitable for measuring and evaluating UDS by autoradiography in primary rat hepatocytes and diploid human fibroblasts and by the LSC approach in diploid human fibroblasts. UDS detection is an appropriate system for inclusion in carcinogenicity and mutagenicity testing programs, because it measures the repair of DNA damage induced by many classes of chemicals over the entire mammalian genome. However, for this system to be utilized effectively, appropriate metabolic activation systems for autoradiographic measurements of UDS in human diploid fibroblasts must be developed, the nature of hepatocyte-to-hepatocyte variability in UDS responses must be determined, and the three suggested protocols must be thoroughly evaluated by using them to test a large number of coded chemicals of known in vivo mutagenicity and carcinogenicity.     

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.     

Repair of ultraviolet radiation damage in xeroderma pigmentosum cells belonging to complementation group F

DNA-repair characteristics of xeroderma pigmentosum belonging to complementation group F were investigated. The cells exhibited an intermediate level of repair as measured in terms of (1) disappearance of T4 endonuclease-V-susceptible sites from DNA, (2) formation of ultraviolet-induced strand breaks in DNA, and (3) ultraviolet-induced unscheduled DNA synthesis during post-irradiation incubation. The impaired ability of XP3YO to perform unscheduled DNA synthesis was restored, to half the normal level, by the concomitant treatment with T4 endonuclease V and ultraviolet-inactivated Sendai virus. It is suggested that xeroderma pigmentosum cells of group F may be defective, at least in part, in the incision step of excision repair.     

X-ray induced sex-chromosome loss, when ring-X chromosome males are irradiated and mated to females carrying mei-9, mei-41 or mei-218

DNA-repair characteristics of xeroderma pigmentosum belonging to complementation group F were investigated. The cells exhibited an intermediate level of repair as measured in terms of (1) disappearance of T4 endonuclease-V-susceptible sites from DNA, (2) formation of ultraviolet-induced strand breaks in DNA, and (3) ultraviolet-induced unscheduled DNA synthesis during post-irradiation incubation. The impaired ability of XP3YO to perform unscheduled DNA synthesis was restored, to half the normal level, by the concomitant treatment with T4 endonuclease V and ultraviolet-inactivated Sendai virus. It is suggested that xeroderma pigmentosum cells of group F may be defective, at least in part, in the incision step of excision repair.     

Cryopreservation and long-term storage of primary rat hepatocytes: Effects on substrate-specific cytochrome P450-dependent activities and unscheduled DNA synthesis

The effects of cryopreservation and long-term storage on substrate-specific cytochrome P45O-dependent activities and unscheduled DNA synthesis were studied in freshly isolated and cryopreserved hepatocytes derived from adult male Fischer 344 and Sprague-Dawley rats. Primary rat hepatocytes were isolated via an in situ collagenase perfusion technique, cryopreserved at –196°C, and thawed at 5 weeks and 104 and 156 weeks post-freezing. In Fischer 344 and Sprague-Dawley rats, cryopreserved hepatocytes were equivalent or similar to freshly isolated hepatocytes in substrate-specific activities for 7-ethoxyresorufin-0-deethylase and dimethylnitrosamine-N-demethylase and unscheduled DNA synthesis responses. No significant differences in activities toward 7-ethoxyresorufin-0-deethylase and dimethylnitrosamine-N-demethylase, the substrate-specific activities for cytochromes P4501A1 and P4501A2 and cytochrome P4502E1, respectively, were observed between freshly isolated and cryopreserved hepatocytes. Similar unscheduled DNA synthesis responses, a measure of DNA damage and repair, were observed after exposure to the genotoxic carcinogens 2-acetylaminofluorene, 7,12-dimethyEbenz[a]anthracene, and dimethylnitrosamine; although some decreases were also observed in Fischer 344 hepatocytes after 104 weeks and Sprague-Dawley hepatocytes after 156 weeks in the highest concentrations tested. These results suggest that cryopreserved hepatocytes, stored for extended periods of time in liquid nitrogen, are metabolically equivalent to freshly isolated hepatocytes in their ability to activate precarcinogens.Abbreviations 2-AAF 2-acetylaminofluorene - DDH₂O distilled deionized water - DMBA 7,12-dimethyIbenz[a]anthracene - DMN dimethylnitrosamine - DMNA dimethylnitrosamine-N-demethylase - DMSO dimethyl sulfoxide - EROD 7-ethoxyresorufin-O-deethylase - F344 Fischer 344 - FBS fetal bovine serum - %IR percentage of cells in repair - LN₂ liquid nitrogen - LSD least significant difference - CG cytoplasmic grains - NNG net nuclear grains - SD Sprague-Dawley - UDS unscheduled DNA synthesis - WE Williams' Medium E     

亚硒酸钠诱发的晶状体上皮细胞DNA损伤及修复

观察了亚硒酸钠（Ｎａ２ＳｅＯ３）在体外作用于大鼠晶状体上皮细胞（ＲＬＥｃｅｌｌｓ）而造成的ＤＮＡ单链断裂（ｓｉｎｇｌｅｓｔｒａｎｄｂｒｅａｋｓ,ＳＳＢ）,并对其ＤＮＡ损伤、修复动力学做了初步研究．发现ＳＳＢ严重程度与亚硒酸钠的浓度呈线性相关,其ＳＳＢ重接修复约在３０～６０ｍｉｎ内完成．还作了有关非程序ＤＮＡ合成（ＵＤＳ）的检测,发现与ＳＳＢ相比,ＵＤＳ发生迟且持续时间更长,提示Ｎａ２ＳｅＯ３可能在体外对大鼠晶状体上皮细胞除造成ＳＳＢ以外,还可能造成其它种类的ＤＮＡ损伤．     

Inhibition of sporulation in Bacillus subtilis by bromodeoxyuridine and the effect on DNA replication

A 5-bromo-2'-deoxyuridine (BUdR)-tolerant derivative of a thymidine (TdR)-requiring strain of Bacillus subtilis was used to examine the effect of BUdR, an analogue of TdR, on sporulation. At a TdR:BUdR ratio which had little effect on growth, sporulation was inhibited if cells were exposed to BUdR during the period of DNA synthesis at the onset of the process. Cells recovered from BUdR inhibition of sporulation if the analogue was removed and DNA replication allowed to continue with TdR alone. BUdR prolonged the period of DNA synthesis during sporulation and experiments with chloramphenicol suggested that this was due in part to unscheduled initiation of new rounds of replication.     

Study of unscheduled DNA synthesis following exposure of human cells to arecoline and extracts of betel nut in vitro.

Aqueous, acetic acid, hydrochloric acid and ethanol extracts of betel nut (Areca catechu L.) have been found to induce unscheduled DNA synthesis in Hep 2 cells obtained from human larynx carcinoma, in vitro. Different concentrations of extracts of betel nut induced dose-dependent unscheduled DNA synthesis in Hep 2 cells. Together with the viability of the Hep 2 cells, our results indicate that the aqueous and acetic acid extracts of betel nut induce relatively more unscheduled DNA synthesis than the hydrochloric acid and ethanol extracts and arecoline. The carcinogenic potency of raw and unprocessed betel nut of North-East India used in this study is discussed.     

Aphidicolin does not inhibit the repair synthesis of mitotic chromosomes

In order to determine the possible role of α-polymerase in repair synthesis, we measured the unscheduled DNA synthesis stimulated by UV-irradiation in mitotic HeLa cells in the presence of Aphidicolin, a specific inhibitor of this enzyme. The results do not show any inhibition of the unscheduled DNA synthesis,indicating that the function of α-polymerase is not essential for repair synthesis of mitotic cells.     

The effect of various inhibitors of DNA synthesis on the repair of DNA photoproducts

The effect on DNA repair of several inhibitors of DNA synthesis has been investigated in CHO cells. Three assays were employed following ultraviolet irradiation of G1 cells: unscheduled DNA synthesis, removal of antibody binding sites and alkaline elution. Cytosine arabinoside and aphidicolin were found to reduce unscheduled DNA synthesis in a dose-dependent manner without affecting the removal of antibody-binding sites. Strand rejoining was also inhibited. These results are consistent with the hypothesis that inhibition is due to premature chain termination during repair synthesis some time after excision of the lesion. Conversely, inhibition of unscheduled DNA synthesis by novobiocin is paralleled by inhibition of excision of the lesion. However, no inhibition of incision was apparent. Since nalidixic acid, an inhibitor of topoisomerase II, did not inhibit excision, it is unlikely that the primary site of action of novobiocin is this topoisomerase. The possibility that a second topoisomerase and/or a polymerase are affected is discussed in the light of previously published data.     

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.