The in vitro unscheduled DNA synthesis (UDS) assay in rat primary hepatocytes: evaluation of 2-furoic acid and 7 drug candidates

The in vitro unscheduled DNA synthesis assay (UDS) is part of the routine genetic toxicology screening at The Upjohn Company. The purpose of this paper is to report results for 8 compounds which were tested in the in-house genetic toxicology program. These compounds represent diverse chemical structure and most of them entered the screening program because they are biologically active in efficacy screens. All tests were carried out under Good Laboratory Practices Regulations of the U.S. Food and Drug Administration. None of the materials reported here produced an increase in UDS and therefore the UDS results with these compounds do not suggest potential for genotoxicity.     

The in vitro unscheduled DNA synthesis (UDS) assay in rat primary hepatocytes: Validation of improved methods for primary culture including data on the lack of effect of ionizing radiation

The in vitro unscheduled DNA synthesis (UDS) assay was evaluated for inclusion in a battery of assays used at The Upjohn Company for evaluation of lead compounds in the development of new and existing drug entities. This evaluation process encompassed aspects of the isolation of hepatocytes and tests of reference mutagens and genotoxins. The flow rate of perfusion solutions and their temperatures were critical in the isolation of high viability hepatocytes in good yield. The attachment of freshly isolated hepatocytes to coverslips was greatly enhanced by coating the coverslips with type III collagen. Results of testing 12 known genotoxic agents (UV light, cyclophosphamide, 7,12-dimethylbenzanthracene, dimethyl-nitrosamine, diethylnitrosamine, 2-acetylaminofluorene, benzo[a]pyrene, methyl methanesulfonate, ethyl methanesulfonate, N-propyl-N′-nitro-N-nitrosoguanidine, benzidine and 4-aminobiphenyl) were in agreement with the literature. The use of X-ray did not induce unscheduled DNA synthesis in hepatocytes. This latter finding draws attention to the inability of this assay to detect agents which result in ‘short-patch’ repair of damage.     

Application of the neutral red assay (NR assay) to monolayer cultures of primary hepatocytes: rapid colorimetric viability determination for the unscheduled DNA synthesis test (UDS)

The neutral red (NR) absorption method was adapted for the determination of cell viability in the UDS assay with primary hepatocyte cultures of the rat. The NR method is rapid, easy to perform, and suitable for handling of large numbers of cultures simultaneously. It can be used for concentration range-finding pre-experiments. In addition, it can easily be integrated into a UDS test protocol for documentation of toxic effects if supplementary cultures for each concentration are established. The time schedule required for the NR assay makes it possible for one person to process the hepatocytes for autoradiography and at the same time determine the toxicity.     

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.     

Characterization of an unscheduled DNA synthesis assay with Syrian hamster embryo cells

The Syrian hamster embryo cell transformation assay is widely used for studies of carcinogenesis. The characterization of an unscheduled DNA synthesis (UDS) assay for these cells is reported. Benzo[a]pyrene, aflatoxin B1 and UV light induced UDS in the cells in a dose-dependent manner without exogenous metabolic activity. Nitrosopiperidine induced UDS as well as gene mutations and cell transformation only in the presence of an exogenous metabolic activation system. The utility of this UDS assay with these cells is discussed.     

Evidences for the function of DNA polymerase-beta in unscheduled DNA synthesis.

The activities of DNA polymerase-alpha and -beta isolated from pig spleen were determined at different temperatures and in the presence of different concentrations of inhibitors. The results were compared with parallel estimations of replicative DNA synthesis and UV-induced repair synthesis in spleen cells. In respect to pCMB and aCTP, polymerase-alpha is more sensitive than polymerase-beta and similarly is replication more sensitive than repair. Repair synthesis and the activity of polymerase-beta decreases at temperatures higher than 40 degrees C whereas both replication and the activity of polymerase-alpha are greatly stimulated at elevated temperatures with optima of 45 degrees C (polymerase-alpha) and 41 degrees C (replication). The results favour the hypothesis that polymerase-beta is involved in repair synthesis.     

Strain differences in in vitro rat hepatocyte unscheduled DNA synthesis (UDS): effect of UV is independent of strain while increased sensitivity is apparent using Fischer-344 instead of Sprague-Dawley rats

The unscheduled DNA synthesis (UDS) assay measures DNA repair following in vitro treatment of rat primary hepatocytes. This report compares the UDS response of primary hepatocytes from 2 widely used rat strains, the Fischer-344 (F344) and Sprague-Dawley (SD) strains. Ultraviolet (UV) light and 5 known genotoxic chemicals were evaluated in each strain in parallel experiments. The chemicals tested were 2-acetylaminofluorene (2-AAF), 4-aminobiphenyl (4-AB), benzidine, dimethylnitrosamine (DMN) and N-propyl-N'-nitro-N-nitrosoguanidine (PNNG). Four of these compounds (2-AAF, 4-AB, benzidine and DMN) require metabolic activation. Benzidine and PNNG were both negative using SD rat hepatocytes, but were weakly positive using F344 rat hepatocytes. In the first of 2 experiments, 4-AB was inconclusive in SD hepatocytes, but strongly positive in F344 cells. In the second experiment, 4-AB was positive in hepatocytes from both strains. 2-AAF was more strongly positive in F344 cells than in SD cells. DMN and UV light induced positive dose responses with little or no differences between strains. It is concluded that hepatocytes from F344 rats may be more sensitive, qualitatively and quantitatively, than hepatocytes from SD rats as indicators of UDS. This difference is not due to intrinsic differences in DNA repair mechanisms but is probably due to differences in drug-metabolizing enzymes between these strains. Thus, for routine screening, F344 rats are preferable for measurement of the in vitro UDS-inducing potential of compounds.     

Testing of hydralazine in in vivo-in vitro hepatocyte assays for UDS and stimulation of replicative DNA synthesis

The vasodilator hydralazine was tested for induction of DNA-repair synthesis and stimulation of replicative DNA synthesis in rat hepatocytes after administration in vivo, either once or repetitively. No increase in unscheduled or replicative DNA synthesis was observed. By contrast, positive controls clearly induced DNA-repair synthesis, either after a single treatment (4-aminobiphenyl, dimethylnitrosamine and methyl methanesulphonate) or after repetitive treatment (benzo[a]pyrene), or stimulated replicative DNA synthesis (carbon tetrachloride and dimethylnitrosamine). Thus, hydralazine displayed no genotoxic and no tumour-promoting activity in these in vivo-in vitro test systems.     

The autoradiographic test for unscheduled DNA synthesis: a sensitive assay for the detection of DNA repair in the HepG2 cell line

We assessed the DNA-repair capacity of HepG2 cells, which were derived from a human hepatoma, by the unscheduled DNA synthesis assay, using the autoradiography protocol (UDS-AR). We evaluated DNA repair following exposure to direct mutagens (4-nitroquinoline-N-oxide (4-NQO), methyl methanesulfonate (MMS)), to mutagens requiring metabolic activation (benzo[a]pyrene (B[a]P), 2-acetylaminofluorene (2-AAF), N-dimethylnitrosoamine (NDMA)) or to structurally related non-mutagens such as pyrene and 4-acetylaminofluorene (4-AAF). All positive compounds tested induced UDS in HepG2 cells. With 4-NQO and MMS, a concentration-dependent increase in net nuclear grains per cell was observed, with 73 and 90% of cells, respectively, in repair at the highest concentration. B[a]P, 2-AAF and NDMA displayed similar dose-dependent UDS responses, but the percentage of cells in repair was lower (about 45%) than that for 4-NQO and MMS. We assessed the genotoxicity of the compounds tested by determining IC(5NNG): the concentration required to induce 5NNG. The compounds studied were ranked in order of IC(5NNG) as follows: 4-NQO = B[a]P > 2-AAF > MMS > NDMA. The UDS assay discriminated between mutagens and non-mutagens, as pyrene and 4-AAF failed to induce DNA repair. The present study demonstrates that UDS can be used as an endpoint for the detection of DNA damage in HepG2 cells.     

Use of cryopreserved hepatocytes for unscheduled DNA synthesis assays

Hepatocytes were isolated from Fischer rats by perfusion and tested for unscheduled DNA synthesis (UDS) induction or cryopreserved for long-term storage at -196 degrees C. Thawed cells could be recovered at greater than 90% viabilities, and were cultured on fibronectin-coated coverslips. The cells attached and spread, and could be used for UDS assessment. Data were compared for fresh and frozen cells from the same animal. Results obtained for net nuclear grains and dose response were similar for the fresh and frozen cells, in response to the carcinogenic compounds methyl methanesulfonate and 7,12-dimethylbenzanthracene, benzo[a]pyrene, and N-methyl-N'-nitro-N-nitrosoguanidine.     

In vitro polyoma DNA synthesis: requirement for cytoplasmic factors.

Purified nuclei from polyoma-infected mouse (3T3) cells were found to be greatly reduced in their ability to synthesize viral DNA in vitro when compared with a crude system consisting of an unfractionated hypotonic lysate of the infected cells. The synthetic capacity of the nuclei could be fully reconstituted when a high-speed cytoplasmic supernatant was added back to them. Cytosols from uninfected mouse, monkey, and hamster cells were equally as effective in stimulating purified nuclei as that of virus-infected mouse cells. Optimal complementation required high concentrations of the cytosol, and most of the complementing activity was destroyed by heating to 60 C. Dialysis had no effect on the activity. Analysis of the viral DNA synthesized in purified nuclei showed an accumulation of Okazaki-type short DNA chains, which could be chased into viral progeny DNA strands if cytosol was added back to the nuclei. Kinetic analysis of the pulse-labeling pattern of viral replicative DNA showed a strong dependence of the extension of viral progeny strands and of the processing of Okazaki-type fragments on the amount of cytosol present during the reaction. It is suggested that the cytoplasmic DNA polymerase might be one of the active components in the cytosol, but most likely not the only one.     

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.