DNA polymerase beta mediates protection of mammalian cells against ganciclovir-induced cytotoxicity and DNA breakage.

The efficacy of suicide herpes simplex virus-1 thymidine kinase (HSVtk)/ganciclovir (GCV) gene therapy is often limited by intrinsic resistance of tumor cells. Here we show that repair of GCV incorporated in DNA is a factor involved in GCV resistance. A protective role of DNA repair in GCV-induced cell killing is supported by the following findings: (a) GCV-exposed Chinese hamster ovary-HSVtk cells exhibited both reduced repair of GCV and cloning efficiency in the presence of a specific polymerase beta (beta-pol) inhibitor, prunasin; (b) DNA beta-pol-deficient mouse fibroblasts were more sensitive to the cytotoxic, apoptosis-inducing, and genotoxic (DNA breakage and chromosomal aberration-inducing) effects of GCV as compared with wild-type and beta-pol-complemented cell lines; (c) methoxyamine, an inhibitor of beta-pol-dependent short-patch base excision repair, sensitized wild-type and complemented beta-pol cells to GCV, whereas it had no effect on the sensitivity of beta-pol-null cells to GCV. Because methoxyamine-mediated sensitization of beta-pol wild-type and beta-pol-complemented cells to GCV did not reach the level of null cells, we suggest that both beta-pol-dependent short- and long-patch base excision repair are involved in protection of cells to GCV. Some implications for HSVtk/GCV gene therapy are being discussed.

Comparison of the genotoxic and apoptosis-inducing properties of ganciclovir and penciclovir in Chinese hamster ovary cells transfected with the thymidine kinase gene of herpes simplex virus-1: implications for gene therapeutic approaches.

We studied the genotoxic and apoptosis-inducing properties of ganciclovir (GCV) and penciclovir (PCV) using Chinese hamster ovary cells stably transfected with the thymidine kinase (tk) gene of herpes simplex virus-1 (HSV-1). Cells expressing HSVtk were 300 and 100 times more sensitive than their isogenic HSVtk- counterparts to the cytotoxic effects of GCV and PCV, respectively. Using radiolabeled drugs, GCV was found to be incorporated into the genomic DNA much more effectively than PCV. GCV was highly potent in inducing chromosomal aberrations compared with PCV, which provoked less sister chromatid exchanges and chromosomal changes using equimolar or equitoxic doses. For both agents, apoptosis was shown to be the major route of cell killing. Time course experiments revealed that neither genotoxicity nor apoptosis were induced within the cell cycle exposed to the drug; they are late events provoked in the following cell cycle(s). This indicates that the incorporation/exposure step of GCV or PCV into DNA is not decisive for triggering genotoxicity and apoptosis, but that events occurring subsequently, presumably during replication of a DNA containing the nucleotide analogs, are of major importance. Because PCV, unlike GCV, induced highly effectively apoptosis without exerting much genotoxicity, the use of PCV as a relatively safe alternative drug for suicide gene therapy of malignant diseases is recommended.

Methylnitrosophenylurea--a highly carcinogenic compound.

In vitro, 1-methyl-1-nitroso-3-phenylurea (MNPU) was easily formed from 1-methyl-3-phenylurea (MPU) and sodium nitrite in an acid environment. In rats MNPU showed a strong carcinogenic effect, inducing tumors of the forestomach. MNPU was formed endogenously after combined administration of MPU and nitrite to rats, as indicated by the induction of forestomach tumors. MPU itself was not carcinogenic. In the metabolism of the herbicide 1,1-dimethyl-3-phenylurea (fenuron) MPU resulted as a demethylation product. The possible significance for preventive oncology and the role of MNPU as an environmental carcinogen are briefly discussed.

Genetic risks of antiviral nucleoside analogues--a survey.

The available informations on the genotoxic effects in experimental systems of the antiherpesvirus nucleosides aciclovir, penciclovir, ganciclovir, brivudine and cidofovir as well as of the antiretrovirals zidovudine (AZT), lamivudine, zalcitabine (ddC), didanosine and stavudine are reviewed. Furthermore, data on carcinogenic activity of these drugs in laboratory rodents are compiled. Most nucleoside analogue antivirals induce chromosomal aberrations but are inactive in gene mutation assays. Carcinogenicity findings in mice and rats are variable but clearly positive for AZT and ddC. The possible mechanisms by which these agents may cause damage in the genetic information are still largely hypothetical, and experimental findings do not permit relevant extrapolations to the situation in man. There is no conclusive evidence that any of the drugs caused tumours in humans. The use of nucleoside analogues in antiviral therapy remains a pragmatic option that seems justified by risk/benefit assessment.

Cytogenetic genotoxicity of antiherpes virostatics in Chinese hamster V79-E cells. I. Purine nucleoside analogues.

The antiherpes virostatics acyclovir (ACV), valaciclovir (VACV), penciclovir (PCV), famciclovir (FCV) and ganciclovir (GCV), which belong to the group of purine acyclic nucleoside analogues, were tested for clastogenic and sister chromatid exchange (SCE)-inducing activity in Chinese hamster V79-E cells upon chronic application with and without a recovery period. ACV induced borderline effects in both cytogenetic assays, a dose-dependent reduction of the mitotic index and an increasing cell cycle delay. With VACV and PCV only a decrease of the mitotic index and an increase of cell cycle delay were observed. FCV was negative with respect to the four parameters studied, presumably due to the incapacity of the target cells of metabolizing FCV to PCV. GCV was a very potent genotoxin in both assays. It induced a statistically significant SCE response even in the range of the cytomegalovirus IC50 of < 10 microM. By variation of the experimental protocol it was shown that SCEs are induced in the second cell cycle following exposure to GCV but not in the first one. It is assumed that the drugs under study are metabolized to their respective triphosphates and then inhibit DNA replication as detected by decreasing mitotic index and increasing cell cycle delay. In the case of GCV it is suggested that GCV-TP is incorporated into the target cell DNA and that chromosomal aberrations and SCEs are secondary lesions due to repair processes at the substituted template.

Incorporation in liposomes as a method for the application of genotoxins of low water solubility in the SCE assay.

Benzo[alpha]pyrene BaP), freely dissolved or incorporated in liposomes prepared from egg yolk lecithin, was checked for SCE induction in Chinese hamster V79-E and rat liver RL-19 cells in vitro. SCE induction in V79-E was observed only when freely dissolved BaP was added together with S9 mix. RL-19 cells were per se highly sensitive to SCE induction by BaP either freely dissolved or incorporated in liposomes. It is suggested that the incorporation of genotoxins in liposomes is a practicable method for the application, in mammalian genotoxicity assays, of agents which are barely soluble or completely insoluble in water, provided no exogenous metabolizing system is required.

Activity of citrinin metabolized by rat and human microsome fractions in clastogenicity and SCE assays on Chinese hamster V79-E cells.

The mycotoxin citrinin is a potent inducer of chromosomal aberrations in the clastogenicity assay on V79-E cells when metabolized by rat and human liver microsomes. Rat and human liver microsomes, standardized on protein content, activate citrinin at equal levels. 5 X 10(-4) M citrinin induces complex translocations in a high frequency as well as defects of chromosomal coiling. Higher concentrations are cytotoxic, lower ones are almost inactive. After metabolization of mycotoxin by rat-kidney microsomes or an S9 mix fraction containing rat liver and kidney microsomes, toxic effects predominate and chromosomal aberrations are diminished. Clastogenic citrinin concentrations do not induce an increase of SCE frequency. Although the mode of action of this mycotoxin on chromosomal structure remains obscure, possible explanations are discussed.

Studies by means of the SCE assay in V79-E Chinese hamster cells on the mode of action of tri-substituted nitrosoureas.

The genotoxic activity of 3,3-diethyl-1-methyl-1-nitrosourea ( DEMNU ), 1,3-dimethyl-3-phenyl-1-nitrosourea ( DMPNU ) and 1-chloroethyl-3-methyl-3-phenyl-1-nitrosourea ( CEMPNU ) was studied in the SCE assay in V79-E cells in vitro. These compounds are very stable in aqueous solutions, but are directly acting genotoxins . The SCE rates increase linearly with the length of the incubation period. This direct activity is presumably due to an intracellular catalytic decomposition. Whereas the SCE-inducing effect of DMPNU and CEMPNU is not influenced by addition of S9 mix, that of DEMNU is strongly potentiated by rat and Syrian hamster S9 mix. This DEMNU activation is an NADPH-dependent enzymatic reaction and is inducible by phenobarbital. The absence of a direct mutagenic effect of DEMNU in the Ames test, as reported by other authors, is probably caused by a striking insensitivity to tri-substituted nitrosoureas of the Salmonella assay. This assumption was substantiated by long-term application of very low DMPNU doses to V79-E. Long-term simultaneous treatment with DMPNU and bromodeoxyuridine (BUdR) significantly diminished the rate of SCE induction.

Patulin, a further clastogenic mycotoxin, is negative in the SCE assay in Chinese hamster V79-E cells in vitro.

Patulin is a potent inducer of chromatid-type aberrations in Chinese hamster V79-E cells, but loses its activity when 9000 g supernatant of rat-liver homogenate is added. The narrow dose range of patulin clastogenicity shows a quantitative relationship between absolute amount of mycotoxin applied and the number of indicator cells treated. Within a dose range permitting survival of V79-E, patulin does not induce an increase of the SCE rate. It is suggested that patulin clastogenicity is caused by interaction with chromosomal proteins and that DNA is not the virtual target of this mycotoxin.

Nitrosated urea pesticide metabolites and other nitrosamides. Activity in clastogenicity and SCE assays, and aberration kinetics in Chinese hamster V79-E cells.

The nitrosoureas 1-methyl-1-nitroso-3-phenylurea, 1-ethyl-1-nitroso-3-phenylurea, 1-methyl-1-nitroso-3-(p-fluorophenyl)urea, 1-methyl-1-nitroso-3-(p-chlorophenyl)urea, and 1-methyl-1-nitroso-3-(p-bromophenyl)urea, as well as their non-nitrosated parent compounds, were checked for induction of chromosomal aberrations and sister-chromatid exchanges in V79-E cells without metabolic activation in vitro. For comparison, methylnitrosourea, ethylnitrosourea and nitrosocarbaryl were included in this study. Whereas the non-nitrosated agents were inactive, the nitroso derivatives were potent clastogens and inducers of SCEs. Clastogenicity parallels SCE induction, but the latter assay is about 10 times more sensitive (based on concentration of substance) than the clastogenicity assay. The dependence of aberration frequency on sampling time, which was studied for 5 nitroso compounds, revealed striking differences. As demonstrated by differential chromatid staining, the lag phase until maximal aberration rates may cover more than 2 cell cycles. Preventive oncological aspects of these nitrosamides and the mechanism of aberration kinetics are discussed.

Use of human-liver microsomes from kidney-transplant donors for the induction of chromatid aberrations and sister-chromatid exchanges by means of pre-carcinogens in Chinese hamster cells in vitro.

Samples of two human livers taken during operation of kidney donor patients were processed for microsome fractions and used for metabolization of cyclophosphamide (CP) and dimethylnitrosamine (DMN) in combination with the NADPH-generating system. Rat-liver microsomes were checked for comparison. Induction of chromatid aberrations and sister-chromatid exchanges in a newly isolated clone of Chinese hamster fibroblasts served as indicators of activity. Human S-9 fractions standardized on protein content showed strong variations of CP and DMN activation. Whereas liver microsomes of one patient (who also suffered from Gaucher's disease) were highly active for both pre-carcinogens and metabolized DMN at the same level as the uninduced rat-liver microsomes, the S-9 fraction from the second patient failed to activate CP, but was distinctly positive for DMN. It is suggested that samples of liver and other organs of renal transplant donors might be a practicable source of freshly prepared human microsome fractions usable in biochemical, genetic and carcinogenetic studies. Problems concerning the extrapolation of results are discussed.

Establishment and cidofovir sensitivity of a cell line from a heart transplant recipient with multiple cutaneous tumors.

A new cell line, designated as Tuwei00, is described. It originated from an Epstein-Barr virus-positive skin tumor biopsy of a heart transplant recipient, whose numerous cutaneous neoplasms were treated with the antiviral drug cidofovir what caused at least transient remissions. The cell line was established in vitro and maintained for more than 70 passages. Cells of early passages were characterized by a slower growth, the inability to form colonies and a higher sensitivity to cidofovir. After overcoming a crisis, the cells grew faster, to a higher density and were able to form adherent colonies from single cells as well as colonies in soft agar. Chromosome analysis showed diploidy/hyperdiploidy at the earlier and hypodiploidy at the later passages. Sensitivity to cidofovir was distinctly higher in early passages of Tuwei00 cells than in later passages and was characterized by distinct decline of cell survival after long term cidofovir exposure. Established normal human keratinocytes, HaCaT cells, which were checked for comparison, showed a low cidofovir sensitivity similar to late passage Tuwei00 cells.

Clastogenicity and sister chromatid exchange induction by ftorafur.

The main effect of Ftorafur at the chromosomal level is the induction of chromatid and chromosome breaks, which is some pronounced in neoplastic or transformed cells than in normal cells. Different cell lines used in the study exhibited both in vitro and in vivo varying sensitivity to Ftorafur. Ftorafur does not increase the frequency of SCE.

Interindividual variation of carcinogen activation by human liver homogenates. A study using dimethylnitrosamine (DMN) and cyclophosphamide (CP) as precursor genotoxic agents and clastogenicity and induction of sister chromatid exchanges in Chinese hamster V79-E cells as endpoints.

9000 g supernatants of liver homogenates from 6 kidney transplant donors were checked in combination with Chinese hamster V79-E cells in vitro for their capacity to activate DMN and CP. 9000 g fractions were standardized on protein content. Induction of chromatid aberrations (clastogenicity assay) and sister chromatid exchanges (SCE assay) served as parameters. DMN activation showed a 1.3-fold inter individual variation and was in the same order of magnitude as that observed with rat liver 9000 g fractions. Striking interindividual differences were found when CP was applied as reference precarcinogen. Most of the samples had a distinctly lower activity than that of the rat but reached, in one case, a similar level. Methodological problems and limitations of genotoxicity tests for precarcinogen screening with respect to extrapolation of results to man are discussed.

The mechanism of cytogenetic genotoxicity of exogenous glutathione in V-79 cells in vitro--implication of hydrogen peroxide and general traits of oxidative chromosome damage.

The mechanism of cytogenetic genotoxicity (clastogenicity, induction, cell cycle delay) of 10(-3) M glutathione in V79-E cells, as described by Thust and Bach (1985), was studied in detail by using different treatment conditions. It was found that 1-cystine is the essential cofactor in the incubation system. Catalase, but not superoxide dismutase, abolished the genotoxic effect, and the iron chelator desferoxamine, as well as the hydroxyl radical scavenger mannitol, diminished the activity. It is suggested that glutathione, in combination with V79-E cells and cystine, forms a hydrogen peroxide-generating system which provokes the adverse effects. Glutathione as well as 1-cysteine and 2-mercaptopropionylglycine, which were checked for comparison, show a "paradoxic genotoxicity," i.e., at 10(-2) M the effects return almost to the level of controls. Concentration dependence and other criteria of cytogenetic genotoxicity observed with glutathione show obvious similarities to those of other oxidatively acting agents and reveal striking differences to the cytogenetic effects of "typical" genotoxins.

[Carcinogen screening in mammalian cells in vitro. The clastogenicity and the SCE test]. / Kanzerogenscreening in Säugetierzellen in vitro. Der Klastogenitäts- und der SCE-Test.

A survey is given about problems related to cheking for genotoxicity of environmental chemicals. Cytogenetic effects, induction of structural chromosome aberrations (clastogenicity) and sister chromatid exchanges (SCEs), are very sensitive indicators of genetic damage in eukaryotic cells. Problems and methods of the clastogenicity and SCE assay in mammalian cells in vitro are described. These bioassays are correlation tests and do not allow a definitive prediction of carcinogenic activity of chemicals, but they are a valuable tool for the evaluation of genetic/carcinogenic risks. Problems of extrapolation to man are discussed.

Cytogenetic properties of Chinese hamster V79-E cells: G-banding, C-banding, nucleolar organizer regions, and sister chromatid exchanges.

A line of the Chinese hamster V79 strain, denominated as V79-E, is cytogenetically characterized. It has a modal chromosome number of 22. Chromosome morphology, G- and C-banding reveal strong differences from the normal complement of the Chinese hamster presumably caused by rearrangements of chromosome segments during the past 20 years of in vitro culture. 4 chromosomes possess terminal nucleolar organizer regions. Spontaneous sister chromatid exchanges occur with a frequency of 0.38 sister chromatid exchanges per chromosome.

[Long-term cultures of experimental CNS tumours as models in neurooncological research (author's transl)]. / Langzeitkulturen experimenteller Tumoren des Zentralnervensystems als Modelle für neuroonkologische Forschungen

Experiences with the behaviour of long-term cultures of experimental CNS tumours of rats induced by methylnitosourea of ethylnitrosourea, resp., are reported. Differences in cell kinetic parameters result in a selection of the best adapted cell type among those ones existing in the primary tumour. In some long-term cultures of gliomas and neurinomas an increasing polymorphism is observed by which these lines gain the morphological appearance of a glioblastoma multiforme. The loss of cell-specific morphological differentiations is a further type of anaplastic changes. In neuroectodermal tumour strains these alterations are temporarily reversible by means of addition of dibutyryl cyclic AMP to the culture fluid. There is no relationship between the type of tumour and the degree of anaplastic changes in vitro. Some cell strains remain constant for years and retain their specific properties. Their application as tester strains in neurooncological studies is recommended.

Screening of potential chemical carcinogens by means of mammalian cells in vitro.

A survey is presented about the current state of the efforts to the development of rapid screening systems for carcinogenic substances by means of mammalian cells in vitro. At present two fundamental ways for the realization of this task seem possible: a) So-called host-mediated assays which are a combination of carcinogen application into intact animals (possibly pregnant animals) and succeeding explantation of organs from these animals or establishment of cell cultures from fetuses following transplacental action of the compound tested. In this type of experiment the metabolization of the compound is accomplished within the organism and the critical problem of metabolic competence of cultured cells is bypassed. b) Direct application of the carcinogen into cell cultures. Beside the hitherto most used fresh embryonic cells there is an increasing tendency to use established cell lines with a rigid post-confluence inhibition of proliferation and an extremely low background of spontaneous alteration in vitro. Possibilities for the metabolic activation in this system are pointed out. The value of "indicators" of carcinogen-induced alterations in relation to neoplastic properties is discussed. Most of the higherto existing test systems are problematic in their relevancy, but the findings published in literature indicate that cell cultures offer a very promissing possibility for the early detection of carcinogenic agents in human environment.

Exogenous glutathione induces sister chromatid exchanges, clastogenicity and endoreduplication in V79-E Chinese hamster cells.

Glutathione (GSH) dissolved in Eagle's MEM and added to cultures of V79-E cells in concentrations between 2.5 X 10(-4) and 10(-3) moles/l for 1 h induces a dose-dependent cell cycle delay, sister chromatid exchanges and clastogenic damage. 7-8% of the metaphases showed endoreduplication at a recovery phase of 25 and 30 h after treatment with 10(-3) moles/l GSH. Higher concentrations were lethal. The highest tolerated dose corresponds to the intracellular GSH level in V79-E cells. In the same range of concentrations, glutathione disulfide was inactive. Endoreduplication induction by GSH is G2-phase specific and endoreduplication metaphases show a reduced occurrence of single SCEs when extrapolated to the diploid complement. The adverse effects of GSH are independent of the presence of serum in the culture fluid but completely abolished when the treatment is performed in Hank's solution instead of MEM. The mechanism of genotoxicity of exogenous GSH is discussed but, at present, no pertinent explanation can be given.