DNA-repair studies with sodium fluoride: comparative evaluation using density gradient ultracentrifugation and autoradiography

Sodium fluoride (NaF) was assayed for the induction of DNA-repair synthesis in WI-38 human diploid fibroblasts and in primary cultures of rat hepatocytes. DNA-repair synthesis in non-replicating DNA was measured by ultracentrifugation of density-labeled DNA in CsCl gradients. When this method was used, NaF did not induce DNA-repair synthesis in either of these cell types. However, when NaF was assayed for induction of unscheduled DNA synthesis (UDS) in rat hepatocytes by autoradiography, an increased net nuclear grain count was observed. Because the autoradiographic results were not confirmed by density-gradient ultracentrifugation of hepatocyte DNA, which is a more definitive technique, it is doubtful whether the autoradiographic results actually represent DNA-repair synthesis. Modifications of the UDS/autoradiography protocol to include more extensive washing resulted in no UDS response. Published reports (Hellung-Larsen and Klenow, 1969; Srivastava et al., 1981) describe the formation of precipitable complexes of Mg2+, F-, and [3H]thymidine triphosphate which suggests that autoradiographic measurement of UDS may lead to artifacts when testing NaF unless extensive washing of the cultures is employed.     

Induction of unscheduled DNA synthesis in primary cultures of rat, mouse, hamster, monkey, and human hepatocytes

Variation in hepatic metabolism between species may be an important factor in the differences observed in chemical carcinogenesis. We examined 6 chemicals representative of 4 chemical classes in the in vitro hepatocyte DNA repair assay using cells isolated from the Fischer-344 rat, B6C3F1 mouse, Syrian golden hamster, cynomolgus monkey and from human liver. Hepatocytes were isolated by in situ or biopsy liver perfusion and incubated with [3H]-thymidine and the test chemical. Unscheduled DNA synthesis (UDS) was measured as net grains/nucleus (NG) by quantitative autoradiography. Qualitative and quantitative differences in UDS responses were observed for every chemical. Liver cultures isolated from the rat, mouse, hamster, human, and monkey and treated with aflatoxin B1 or dimethylnitrosamine all yielded dose-related increases in NG. Human, rat, and hamster hepatocyte cultures yielded positive responses following exposure to the aromatic amines 2-acetylaminofluorene, 4-aminobiphenyl, and benzidine, whereas cultures isolated from the monkey and mouse yielded less than 0 NG. Treatment with benzo[a]pyrene (BAP) produced strong positive responses in monkey and human hepatocyte cultures, weak positive responses in hamster cultures, and equivocal or negative responses in rat and mouse hepatocyte cultures. Hepatocyte function was assessed by measurement of DNA content, glutathione content, BAP hydroxylase activity, p-nitroanisole-O-demethylase activity, p-nitrophenol conjugation, and urea synthesis rates. The functional capabilities of isolated hamster, monkey, and human hepatocyte cultures do not appear to correlate with UDS responses observed for any compound; however, they indicate that the cultures were metabolically competent at the time of chemical exposure. These studies suggest that rat hepatocytes are a suitable model for human hepatocytes, whereas mouse and male monkey hepatocytes may be insensitive to aromatic amines.     

DNA repair synthesis induced by N-hydroxyurea, acetohydroxamic acid, and N-hydroxyurethane in primary rat hepatocyte cultures: comparative evaluation using the autoradiographic and the bromodeoxyuridine density-shift method

N-Hydroxyurea and two structurally related compounds, acetohydroxamic acid and N-hydroxyurethane, were investigated for their potential to induce DNA repair synthesis in primary rat hepatocyte cultures. Repair was determined as repair replication by means of the bromodeoxyuridine density-shift method and, in the same cell preparations, as unscheduled DNA synthesis (UDS) by autoradiography. For all 3 compounds, a clear concentration-dependent induction of DNA repair replication could be demonstrated. Interpretation of the UDS data, however, depended on the mode whereby the results were evaluated. Expression of the results as net grains per nucleus after subtraction of cytoplasmic from nuclear grain counts yielded statistically significant increases over the control values for all compounds. In contrast, no significant changes of the nuclear labeling were obtained when nuclear and cytoplasmic grain counts were plotted separately. These findings demonstrate that the two modes to present UDS data may lead to different conclusions, a consequence of the uncertainty regarding the origin and importance of the cytoplasmic background. The observation that both hydroxyurea and the structurally related compounds acetohydroxamic acid and N-hydroxyurethane induce DNA repair in primary hepatocyte cultures suggests that metabolism-dependent genotoxicity may be a common property of aliphatic hydroxamic acids.     

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: I. Unscheduled DNA synthesis assay in rat hepatocyte cultures

A protocol based primarily on current laboratory practices in the performance of the unscheduled DNA synthesis (UDS) assay with primary rat hepatocyte cultures has been developed. These guidelines were developed using tabulated responses to a detailed questionnaire completed by North American and European governmental, university and contract laboratories involved with the UDS test. This report identifies those modifications to previously described methodologies which are used on a regular basis and also serves to clarify confusing or inconsistent practices. Although this protocol pertains specifically to the use of primary rat hepatocyte cultures, it can be modified to incorporate other types of cells in which certain aspects remain the same.     

Unscheduled DNA synthesis induced by N-acetoxy-2-acetylaminofluorene is not sensitive to regulation by ADP-ribosyl transferase

We have directly compared in resting human mononuclear leukocytes the DNA repair effects caused by ADP-ribosyl transferase (ADPRT) activity following DNA damage induction by gamma radiation, UV radiation, ethylene oxide (EO) and N-acetoxy-2-acetylaminofluorene (NA-AAF). The presence of inhibitors of ADPRT during the quantitation of unscheduled DNA synthesis (UDS) resulted in about a 2-fold increase of UDS when induced by gamma radiation, UV radiation or EO. The stimulation of UDS by EO, UV- or gamma-radiation in the presence of an ADPRT inhibitor was equally strong whether 1 mM or 10 mM hydroxyurea was used to suppress scheduled DNA synthesis. The level of NA-AAF induced UDS was not affected by inhibitors of ADPRT. In addition, direct estimation of ADPRT activity revealed that at doses giving maximal UDS, NA-AAF damage did not induce a measurable enzymatic activity whereas gamma-radiation, UV radiation and EO all showed a significant dose response increase. We have interpreted our data to mean that NA-AAF induced UDS estimates DNA repair relating mainly to DNA lesions that are recognized with difficulty, and hence, the rate of endonuclease-induced DNA strand break accumulation is not sufficient to allow a stimulation of ADPRT and affect the quantitation of UDS.     

Induction of unscheduled DNA synthesis in cultured human oral keratinocytes by sodium fluoride

The effect of treatment of cultured human oral keratinocytes with sodium fluoride (NaF) has been investigated with respect to induction of unscheduled DNA synthesis (UDS). Oral keratinocytes were isolated from excised buccal mucosa of normal individuals by trypsinization at 4 degrees C overnight, followed by separation of the epithelium of mucosa from lamina propria mucosae with forceps. Isolated cells were cultured in vitro and all experiments were performed with secondary cultures. For detection of UDS, the keratinocytes were cultivated with medium containing 1% fetal calf serum (FCS) for 2 days and then treated with 100-300 micrograms/ml NaF for 4 h in medium containing 1% FCS and 10 mM hydroxyurea (1% FCS-HU medium). Following treatment with NaF, UDS was measured by direct scintillation counting of [3H]thymidine incorporated into DNA of the cells in 1% FCS-HU medium. Significant levels of UDS were induced in a dose-related fashion by NaF treatment. The results suggest that NaF causes DNA damage in cultured human oral keratinocytes.     

Unscheduled DNA synthesis in cultured ataxia telangiectasia fibroblast-like cells

The capacity of cultured fibroblast-like cells from 5 ataxia telangiectasia (AT) patients to repair γ-ray-induced damage formed in DNA under aerobic conditions was measured by unscheduled DNA synthesis (UDS). The AT cultures exhibited generally reduced UDS compared to the normal cultures for γ-ray doses of 4–12 kR. Reduced UDS in the AT cultures was observed for repair-incubation periods at which UDS was not saturated (0.5 and 1 h), and for periods characterized by maximal amounts of UDS in both the normal and AT cultures (2 and 4 h). The results suggest a class of lesions repairable in the normal cells by UDS within 1 h many persist in the AT cells for 4 h or longer.     

Assessement of the relation between the initial viability and the attachment of freshly isolated rat hepatocytes used for the in vivo/in vitro DNA repair assay (UDS)

Crucial steps of the in vivo/in vitro DNA repair assay (UDS) are the hepatocyte isolation procedure and the establishment of the hepatocyte cultures. Since the attachment of the isolated hepatocytes on the surface of the culture vessel is an essential prerequisite for the in vitro part of this assay to yield scorable autoradiograms, we assessed the relation between the initial viabilities of hepatocyte preparations and the resulting attachment efficiencies from 286 rats. The initial viability was determined by means of the trypan blue dye exclusion assay. The actual cell number was corrected for the viability and a constant number of 2.5 × 10⁵ viable cells were seeded into each well of gelatinized six-well dishes. The amount of adherent cells was determined after a 1.5-h attachment period using a recently described modification (Fautz et al., 1991) of the neutral red dye absorption assay. The attachment is described by the optical density at 540 nm obtained after the elution of neutral red from the adherent cells (OD₅₄₀ value).To facilitate a comparison of the data we divided the 286 animals into eight arbitrary viability groups. The mean values of the viability groups were 53.1, 62.2, 66.3, 68.4, 70.9, 73.6, 76.9, and 84.0% living cells. Although there was a great interindividual variation, the resulting mean OD₅₄₀ values were nearly uniform, about 0.5, in all eight groups, regardless of the initial viability of the hepatocytes.UDS data obtained from 46 animals treated with the positive control chemical 2-acetylaminofluorene demonstrated that there was no correlation between the in vitro DNA repair capacity and the initial viability or the attachment efficiency of the hepatocytes.Our results suggest that (i) great interindividual differences exist between the attachment of particular cell preparations with no regard to the initial viability, (ii) the correction of the cell number for viability leads to relatively uniform OD₅₄₀ mean values and (iii) for an in vivo/in vitro UDS assay even cell suspensions with relatively low viabilities can be used since they will yield adherent cultures which are capable of DNA repair synthesis. The latter item often allows a reduction in the number of animals required for this in vivo assay because it is not necessary to perform repeated experiments because of low viability preparations.     

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     

Ethanol inhibits hormone stimulated hepatocyte DNA synthesis

Insulin, glucagon, and epidermal growth factor (EGF) addition stimulated DNA synthesis in primary hepatocyte cell cultures prepared from adult rat liver. The addition of ethanol (20-200mM) to the culture medium resulted in a substantial reduction in DNA synthesis as measured by 3H-thymidine incorporation and autoradiography. This effect was specific for differentiated hepatocytes compared to fibroblasts and two other human hepatoma cell lines. These studies demonstrate in a cell culture system that one of the major properties of ethanol is the inhibition of hepatocyte DNA synthesis.     

Defect in UV-induced unscheduled DNA synthesis in cultured epidermal keratinocytes from xeroderma pigmentosum

DNA repair synthesis in 8 explant-outgrowth cultures of epidermal cells isolated from variant and complementation groups A and E of xeroderma pigmentosum (XP) was examined by measuring unscheduled DNA synthesis (UDS) on autoradiographs. The extents of UDS in XP epidermal cells were compared with those in normal epidermal cells obtained from 26 subjects. In both normal and XP epidermal cells, UDS was induced dose-dependently by radiation at doses of 5-20 J/m2. XP epidermal cells showed various extents of defect in DNA repair depending on the type of XP. In XP-A, the extent of UDS in epidermal cells was very low, being seen in only 3-10% of the normal epidermal cells. But epidermal cells isolated from XP-E and XP-variants exhibited relatively high levels of residual DNA repair; i.e., 69-84% of the control in XP-E and 67-85% in XP-variant. The extents of UDS in XP epidermal cells were almost the same as those in fibroblastic cells isolated from the same specimens.     

Unscheduled DNA synthesis in human hair follicles after in vitro exposure to 11 chemicals: comparison with unscheduled DNA synthesis in rat hepatocytes.

A new method is described to investigate unscheduled DNA synthesis (UDS) in human tissue after exposure in vitro: the human hair follicle. A histological technique was applied to assess cytotoxicity and UDS in the same hair follicle cells. UDS induction was examined for 11 chemicals and the results were compared with literature findings for UDS in rat hepatocytes. Most chemicals inducing UDS in rat hepatocytes raised DNA repair at comparable concentrations in the hair follicle. However, 1 of 9 chemicals that gave a positive response in the rat hepatocyte UDS test, 2-acetylaminofluorene, failed to induce DNA repair in the hair follicle. Metabolizing potential of hair follicle cells was shown in experiments with indirectly acting compounds, i.e., benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine. The results support the conclusion that the test in its present state is valuable as a screening assay for the detection of unscheduled DNA synthesis. Moreover, the use of human tissues may result in a better extrapolation to man.     

Inability of chrysotile asbestos fibers to modulate the 2-acetylaminofluorene-induced UDS in primary cultures of rat hepatocytes

There is now growing evidence that asbestos fibers could act in association with genotoxic compounds, either as cocarcinogens or promoters, in the process of carcinogenesis. The hepatocyte/UDS assay system has been taken to advantage to investigate the capacity of fibers to modulate the effects of genotoxic compounds on the cell, as we previously demonstrated the hepatocytes can engage in phagocytosis of chrysotile fibers. Measurement of UDS was performed by a biochemical procedure involving liquid scintillation counting (LSC) of a purified DNA fraction as well as by radioautography. Both LSC and radioautography revealed that chrysotile asbestos fibers UICC B at concentrations up to 100 micrograms/ml do not elicit UDS, whereas 2-acetylaminofluorene (2-AAF) at low concentrations (0.05-0.625 micrograms/ml) significantly induces it in parallel positive controls. In an attempt to test the cocarcinogen hypothesis, cultures of hepatocytes were simultaneously exposed for 20 h to 2-AAF (0.05 and 0.25 micrograms/ml) and asbestos fibers (1 and 10 micrograms/ml) given as simple mixtures. It was found that the 2-AAF-induced UDS activity was the same whether fibers were present or not. This was observed with both UDS evaluation procedures at all concentration combinations selected. An analysis of variance applied to the data collected from several experiments confirmed that there was no significant 2-AAF-fiber interaction. Our data suggest the absence of intrinsic genotoxic properties for chrysotile fibers. They also indicate that the modulation of the cellular response to genotoxic agents by asbestos fibers is not detected under our test conditions and may require longer-term exposures to be expressed.     

Species differences in cytotoxic and genotoxic effects of phenacetin and paracetamol in primary monolayer cultures of hepatocytes

The cytotoxic and genotoxic effects of phenacetin and paracetamol were examined in monolayer cultures of hepatocytes isolated from the mouse, hamster, rat and guinea pig. No marked increase in unscheduled DNA synthesis (UDS) after exposing hepatocytes from any of the species to phenacetin was observed. At cytotoxic concentrations of paracetamol, an increased UDS in mouse hepatocytes in vitro was observed. Pretreatment of the mice by inducers of drug-metabolizing enzymes, such as 3-methylcholanthrene and Aroclor 1254, lowered the concentration threshold for the toxic responses. With rat hepatocytes only a minor increase in UDS was noted, while with hepatocytes from hamsters and guinea pigs in fact a decrease was seen. The narrow range observed between the cytotoxic and genotoxic effects of paracetamol makes it difficult to predict whether the initial DNA damage could lead to a mutation or whether the cells will die before the mutation is expressed. With respect to the cytotoxic effects, hamster hepatocytes were found to be most susceptible to paracetamol, followed by mouse, while rat and guinea pig were less affected. These data were in accordance with in vivo findings (Davis et al., 1974), indicating the potential value of hepatocyte culture when screening for possible liver toxic substances.     

Characterization of the in vitro unscheduled DNA synthesis assay in primary lung cells of the rat

The in vitro unscheduled DNA synthesis (UDS) assay has been evaluated in rat primary lung cells with known genotoxicants. The autoradiographic method was employed to detect UDS in both alveolar macrophages and primary pulmonary cells. Data of a time course study revealed that a high radioactive labeling of DNA repair was achieved after a 16-h incubation with [3H]thymidine. Coupled with low serum (1%), hydroxyurea at the concentration of 20 mM inhibited regular DNA synthesis in primary lung cells in a satisfactory manner (81-88% inhibition). With this protocol, a dose-related increase in UDS was induced by N-methyl-N'-nitro-N-nitrosoguanidine and 2-aminoanthracene in both rat alveolar macrophages and primary lung cells. The results suggest that primary rat lung cells in culture possess DNA-repair ability and that the UDS assay may be useful for assessing the pulmonary genotoxic effect of chemicals in this cell system.     

Unscheduled DNA synthesis in human hair follicles after in vitro exposure to 11 chemicals: comparison with unscheduled DNA synthesis in rat hepatocytes

A new method is described to investigate unscheduled DNA synthesis (UDS) in human tissue after exposure in vitro: the human hair follicle. A histological technique was applied to assess cytotoxicity and UDS in the same hair follicle cells.UDS induction was examined for 11 chemicals and the results were compared with literature findings for UDS in rat hepatocytes. Most chemicals inducing UDS in rat hepatocytes raised DNA repair at comparable concentrations in the hair follicle. However, 1 of 9 chemicals that gave a positive response in the rat hepatocyte UDS test, 2-acetylaminofluorene, failed to induce DNA repair in the hair follicle.Metabolizing potential of hair follicle cells was shown in experiments with indirectly acting compounds, i.e., benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine.The results support the conclusion that the test in its present state is valuable as a screening assay for the detection of unscheduled DNA synthesis. Moreover, the use of human tissues may result in a better extrapolation to man.     

Effects of intracellular cyclic AMP and cyclic GMP levels on DNA synthesis of young-adult rat hepatocytes in primary culture.

Possible roles of dibutyryladenosine 3',5'-cyclic monophosphate (cAMP) and dibutyryl-guanosine 3',5'-cyclic monophosphate (cGMP) in regulation of hepatocyte DNA synthesis were examined using primary cultures of young-adult rat hepatocytes maintained in arginine-free medium. Throughout the experimental period, nonparenchymal cells were hardly observed in the selective medium. When epidermal growth factor (EGF) was added to the cultures, a transient increase in the intracellular cAMP level preceded the elevation of hepatocyte DNA synthesis. EGF-stimulated hepatocyte DNA synthesis was remarkably enhanced by the elevation of the intracellular cAMP level induced by treatment with cAMP alone or a combination of cAMP and theophylline, an inhibitor of cyclic nucleotide phosphodiesterase. Furthermore, the early elevation of intracellular cAMP alone, which was induced by treatment with the combination of cAMP and theophylline, caused a remarkable increase in hepatocyte DNA synthesis. On the other hand, addition of EGF to the cultures caused a rapid decrease in the intracellular cGMP level followed by an increase in hepatocyte DNA synthesis. EGF-stimulated hepatocyte DNA synthesis was severely suppressed or completely inhibited by the elevation of the intracellular cGMP level induced by treatment with cGMP alone or a combination of cGMP and dipyridamole, a specific inhibitor of cGMP phosphodiesterase. These findings indicate that cAMP and cGMP act oppositely on the regulation of DNA synthesis of young-adult rat hepatocytes in primary culture: cAMP plays a positive role, whereas cGMP plays a negative role. Also it is strongly suggested that an early elevation of the intracellular cAMP level is essential for the onset of DNA synthesis in hepatocyte primary cultures.     

Regulation of the proliferation of primary rat hepatocytes by eicosanoids

DNA synthesis in primary adult rat hepatocyte cultures was promoted by epidermal growth factor (EGF), arachidonic acid, and prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha). Growth promotion by EGF was blocked by 0.1 mM indomethacin and 1 mM aspirin, without affecting cell viability. If verapamil was present in the medium when EGF was added, the growth response was inhibited. Hepatocytes stimulated by EGF or arachidonic acid released PGE2 and PGF2 alpha into the culture medium. This was diminished if 0.1 mM indomethacin was also in the medium. The importance of autocrine regulation of hepatocyte growth by prostaglandins is discussed.     

Unscheduled DNA synthesis in rat tracheal epithelial cells, hepatocytes and spermatocytes following exposure to methyl chloride in vitro and in vivo

Measurement of DNA repair as unscheduled DNA synthesis (UDS) in vitro following exposure in vivo in multiple tissues from the same treated animal can provide valuable information relating to the tissue- and organ-specificity of chemically induced DNA damage. UDS was evaluated in primary cultures of rat tracheal epithelial cells, hepatocytes and pachytene spermatocytes after exposure in vitro to methyl chloride (MeCl), and after isolation from the same treated animal following inhalation exposure in vivo. Concentrations of 1-10% MeCl in vitro induced UDS in hepatocytes and spermatocytes, but not in tracheal epithelial cells. Inhalation exposure to MeCl in vivo (3000-3500 ppm 6 h/day for 5 successive days) failed to induce DNA repair in any cell type. In vivo exposure to 15 000 ppm MeCl for 3 h also failed to induce UDS in tracheal epithelial cells and spermatocytes, but did cause a marginal increase in UDS in hepatocytes. Thus, MeCl appears to be a weak, direct-acting genotoxicant. While activity could be measured in hepatocytes and spermatocytes directly in vitro, only extremely high concentrations of MeCl elicited a response in the whole animal, and then only in hepatocytes.     

Comparative induction of unscheduled DNA synthesis by physical and chemical agents in non-proliferating primary cultures of rat hepatocytes

The incorporation of [³H]thymidine into DNA due to unscheduled DNA synthesis (UDS) induced by N-OH-2-acetylaminofluorene (N-OH-AAF), aflatoxin B₁ (AFB₁), ethyl methanesulfonate (EMS) and ultra-violet light was quantitated by autoradiography and by scintillation spectrometry on acid precipitable macromolecules or DNA insolated by isopycnic banding in cesium chloride (CsCl). Dose-dependent increases in UDS due to N-OH-AAF and AFB₁ treatment were found. Only 2-fold increases at the highest dose levels were found, however, when incorporated [³H]thymidine was quantitated by scintillation spectrometry. Seven, 11, and 25-fold increases in UDS induced by AFB₁, N-OH-AAF and ultra-violet light, respectively, were found when incorporated [³H]thymidine was quantitated by autoradiography, indicating a high sensitivity for detecting ‘long patch’ repair by this technique. Scintillation spectrometry was completely ineffective in detecting EMS-induced UDS, whereas autoradiography demonstrated a small, but significant induction in [³H]thymidine incorporation at high dose levels. The non-proliferative nature of the primary hepatocyte prohibits the uniform radioactive prelabeling of DNA, necessary in other techniques, for the detection of ‘short patch’ repair induced by compounds such as EMS. Therefore, the sensitivity of the primary cultured rat hepatocyte in conjunction with UDS for detecting DNA damage caused by mutagens and carcinogens which induce ‘short patch’ repair may be limited to the autoradiographic analysis of the unscheduled incorporation of [³H]thymidine.