Genotoxicity of carcinogenic N-nitrosopropylamine derivatives in the hepatocyte primary culture/DNA-repair test

The genotoxicity of N-nitrosodipropylamine, 8 of its oxidized derivatives and N-nitroso-2,6-dimethylmorpholine was examined in the hepatocyte primary culture (HPC)/DNA repair test. Nine N-nitrosamines which are known to be carcinogenic and mutagenic were clearly positive in the HPC/DNA-repair test. N-Nitroso(2,3-dihydroxypropyl) (2-hydroxypropyl)amine did not elicit DNA repair, but showed a borderline mutagenic response in the Salmonella/microsome test. Thus, the HPC/DNA-repair test displays a comparable capacity to the bacterial mutagenesis test for detecting the genotoxic effects of this class of carcinogens.     

Genotoxicity of quinone pigments from pathogenic fungi

The genotoxicity and mutagenicity of several kinds of quinone pigments from pathogenic fungi were examined by means of the hepatocyte primary culture (HPC)/DNA repair test and of Ames test with TA98 and TA100. Clear genotoxicity of the two quinone chemicals, xanthomegnin and luteosporin were observed in the HPC/DNA repair test, though definite mutagenicity was not detected in the Salmonella microsome test. These two pigments are thus suspected to be genotoxic carcinogens.     

Measurement of genotoxic activity in multiple tissues following inhalation exposure to dimethylnitrosamine

Chemically-induced DNA repair was measured as unscheduled DNA synthesis (UDS) in selected tissues isolated from rats following in vivo exposure to inhaled dimethylnitrosamine (DMN). UDS was evaluated in epithelial cells from rat nasal turbinates and trachea, in hepatocytes and in pachytene spermatocytes from the same treated animal. At nominal concentrations of 500 and 1000 ppm of DMN in air, chemically-induced DNA repair was observed in the epithelial cells of the upper respiratory system. DMN also entered the circulation, as evidenced by a strong DNA-repair response in hepatocytes. No DNA repair was observed in pachytene spermatocytes indicating either that DMN or its active metabolites did not reach the testes in sufficient concentration to induce DNA repair or that the testes lacked the capability to metabolically activate the compound. These results illustrate the potential of this approach to assess the organ-specific genotoxicity of environmental chemicals.     

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.     

Genotoxicity of pyrene oxide and 1-nitropyrene oxides in hepatocyte primary culture/DNA repair test

The genotoxicity of a pyrene oxide, 1-nitropyrene (NP) oxides and other related compounds was examined in the hepatocyte primary culture (HPC)/DNA repair test. Pyrene 4,5-oxide and both 1-NP-4,5-oxide and 1-NP-9,10-oxide elicited clearly positive responses of DNA repair. In this assay, 1-NP itself was weakly positive. However, other related chemicals such as pyrene, 1-nitro-3-hydroxypyrene, 1-nitro-6-hydroxypyrene, and 1-nitro-8-hydroxypyrene did not generate positive responses.     

Excision repair in permeable arrested human skin fibroblasts damaged by UV (254 nm) radiation: evidence that alpha- and beta-polymerases act sequentially at the repolymerisation step

We have characterised far-ultraviolet-radiation-induced DNA-repair synthesis in permeabilised arrested (non-dividing) primary human skin fibroblasts. Approximately half the maximum repair synthesis is seen after a UV fluence of 4.0 Jm-2 and little additional incorporation was observed at fluences above 20.0 Jm-2. UV-damaged permeable cells were treated with specific inhibitors of DNA polymerase alpha and beta, both alone and in combination. The degree of inhibition of repair incorporation by aphidicolin indicates that polymerase alpha is involved in the majority (85-90%) of repair synthesis after both high and low (less than 4.0 Jm-2) UV fluences. Dideoxythymidine triphosphate seems able to inhibit DNA-repair synthesis only when polymerase alpha is fully or almost fully functional, indicating that polymerase beta is unable to substitute in repair for an alpha polymerase blocked by aphidicolin. These data suggest that the two enzymes may act sequentially to complete repair patches rather than acting independently.     

DNA damage in HeLa S3 cells by an antitumor drug ledakrin and other antitumor 1-nitro-9-aminoacridines

Ledakrin and seven other antitumor and cytotoxic derivatives of 1-nitro-9-aminoacridine were shown to induce DNA-single strand breaks in HeLa S3 cells as found by alkaline sucrose gradient centrifugation. The induced DNA damage is of non-random character. Some of Ledakrin-induced DNA breaks are probably generated by endonucleolytic cleavage in the course of repair processes as indicated by experiments with Novobiocin, an antibiotic preventing the incision step of DNA repair. Other Ledakrin-induced DNA breaks observed on alkaline sucrose gradients may arise from alkali-labile sites in DNA. Most of such sites seem to be converted to breaks after brief exposure to alkali. The extent of DNA damage by 1-nitro-9-aminoacridines was found to be correlated with cytotoxic activities of these compounds against HeLa S3 cells. Furthermore, Ledakrin and other derivatives seem to induce DNA-repair synthesis in HeLa S3 cells as judged by the stimulation of hydroxyurea (HU)-resistant incorporation of [³H] thymidine into DNA. The agents studied differ in their concentrations required to produce a considerable stimulation of DNA repair, whereas the maximal level of this effect is similar for all the derivatives assayed. The former values are correlated with cytotoxic activites of these compounds and seem to reflect the overall extent of DNA damage by 1-nitro-9-aminoacridines. Stimulation of DNA-repair synthesis is gradually shut off during prolonged incubation of the cells with Ledakrin or during postincubation of the cells in a drug-free medium. Such postincubation results also in the gradual accumulation of DNA-single strand breaks as observed by alkaline sucrose centrifugation. Hence, HeLa S3 cells are incapable of efficiently removing DNA damage by 1-nitro-9-aminoacridines, though the drug's action activates temporarily some repair mechanisms.The reported results suggest that overall DNA damage may contribute to the cytotoxic effects of 1-nitro-9-aminoacridines besides previously found ability of these agents to form interstrand DNA cross-links.     

The hepatocyte primary culture/DNA repair test using hepatocytes from several species

The hepatocyte primary culture/ DNA repair test, originally validated with rat hepatocytes, has been extended to use hepatocytes from other species including mouse, hamster, guinea pig, rabbit, monkey and human. Both qualitative and quantitative differences have been observed when chemicals are examined in the hepatocyte primary culture/DNA repair test using hepatocytes from more than one species. Examples are discussed that illustrate that the genotoxicity of a chemical can be a species-specific response and that multi-species testing permits a more complete assessment of genotoxicity.Abbreviations HPC hepatocyte primary culture - MOCA 4,4-methylene-bis-2-chloroaniline     

Structure-activity relationships in the rat hepatocyte DNA-repair test for 300 chemicals

312 chemicals/mixtures were tested for genotoxicity in the rat hepatocyte/DNA-repair test. A variety of structure-activity relationships was evident. Of the 309 pure chemicals, 142 were positive. Of these, 43 were judged by IARC to have sufficient or limited evidence of carcinogenicity and none of the remainder was a proven noncarcinogen. Among the 167 negative chemicals, 44 were carcinogens. Some of these are known to be genotoxic in other systems, but based on several lines of evidence, many are considered to be epigenetic carcinogens that lack the ability to react with DNA and rather lead to neoplasia by nongenotoxic mechanisms.     

Methodology for the testing of food dyes for genotoxic activity: experiments with red 2G (C.I. 18050).

A methodology for investigating genotoxicity of food colours using the fluctuation and DNA-repair assays with bacteria is described. In addition, a liquid repair test, developed to permit incorporation of microsomes and the quantitative estimation of cell viability, has been characterised with a number of positive control agents. Results obtained in these systems suggest that the food colour Red 2G induces repairable DNA damage and base-substitution mutation, but only in the presence of a rat-liver microsomal preparation. The significance of the data in the light of other toxicological information is discussed.     

Chromosomal aberrations and micronuclei induced in rat and mouse bone marrow cells by sodium nitrate

The genotoxicity of several anthraquinone compounds metabolically related to aflatoxin B1 was examined by means of the hepatocyte primary culture (HPC)/DNA repair test and the Salmonella microsome mutagenesis test, and compared to versicolorins A and B which are potent mutagenic and genotoxic intermediates of the aflatoxin biosynthetic pathway. 6,8-O-Dimethyl-versicolorins A, B and 6-deoxyversicolorin A were found to be strongly mutagenic and genotoxic. Genotoxicity of versicolorin A and 6,8-O-dimethylversicolorin A was stronger than that of versicolorin B and 6,8-O-dimethylversicolorin B, respectively, in the HPC/DNA repair test. Nidurufin and norsolorinic acid, which do not possess a bisfuran ring, exhibited questionable activities for mutagenicity and no genotoxicity. It is suspected that 6,8-O-dimethylversicolorins A, B and 6-deoxyversicolorin A as well as versicolorins A and B are genotoxic carcinogens.     

DNA repair synthesis in guinea pig pancreatic slices following in vitro exposure to N-nitrosomethylurethane.

The incorporation of thymidine into DNA in the presence of hydroxyurea (HU) by guinea pig pancreatic slices following exposure to N-nitrosomethylurethane (NMUT) was used to follow DNA repair synthesis. HU was used to suppress normal replicative DNA synthesis. Slices from the duodenal segment of the pancreas were exposed for periods of 15 to 90 min to NMUT at concentrations of 2 to 20 mM, then incubated in tritiated thymidine ([H3]-TdR) free of carcinogen, and radioactivity in DNA was determined. NMUT induced a a dose- and time-dependent increase in HU-insensitive thymidine incorporation. This stimulated incorporation, which could be attributed to repair synthesis, occurred immediately following the treatment and was largely complete within 3 h.     

Comparison of 7 azo dyes and their azo reduction products in the rat and hamster hepatocyte primary culture/DNA-repair assays

Pursuant to the characterization of species differences in the effects of chemical carcinogens, several studies have demonstrated that hamster hepatocytes are more effective than rat hepatocytes in mediating the metabolic activation of certain chemicals to their genotoxic (i.e., mutagenic) derivatives. In the present investigation, a comparison of the amount of DNA repair induced in rat and hamster hepatocytes by 7 azo dyes and 7 aromatic amine azo reduction products of the dyes was performed using the primary hepatocyte culture/DNA repair (HPC/DR) assay. Congo Red and its azo reduction product, benzidine, were more potent inducers of DNA repair in hamster than in rat hepatocytes, whereas Trypan Blue and its reduction product, o-tolidine, were equipotent in the 2 hepatocyte systems. Evans Blue, another o-tolidine-based dye, elicited a greater DNA-repair response in hamster hepatocytes. The absolute potency of these dyes, however, was much less than their reduction products. o-Aminoazotoluene was the most potent of the dyes tested, and its DNA repair-inducing activity was much greater than that of its azo reduction products, o-toluidine and 2,5-diaminotoluene. Ponceau SX, which is carcinogenic in hamsters, but not in rats, was inactive in both hepatocyte systems. Dimethylaminobenzeneazo-1-naphthalene and its 2-naphthalene congener, as well as the 1- and 2-naphthylamine azo reduction products of these dyes, were more potent in hamster than in rat hepatocytes. However, the DNA repair-inducing activities of the parent dyes could not be entirely accounted for by the potencies of their respective naphthylamine derivatives. Taken together, these findings extend previous observations of the superior metabolic activation capabilities of hamster, relative to rat hepatocytes, and further demonstrate the utility of testing chemicals in both the hamster and rat HPC/DR assays.     

Effects of DNA replication inhibitors on UV excision repair in synchronised human cells.

When HeLa cells are irradiated with UV and treated with the DNA synthesis inhibitors hydroxyurea (HU) and 1-beta-D-arabinofuranosylcytosine (ara C), DNA strand breaks accumulate at sites where excision repair of DNA damage has been inhibited after the incision step. This break accumulation occurs in mitotic, G1 and S phase cells. But UV-induced repair synthesis of DNA, as measured by [3H]thymidine incorporation into unreplicated DNA, is not inhibited by HU and ara C in G1 or S phase cells, even though replicative synthesis is virtually abolished. Repair and replication must therefore utilise different DNA precursor pools, or different DNA synthetic systems; and the action of Hu and ara C in causing strand break accumulation may occur at the ligation step of excision repair.     

Effect of 3-aminobenzamide on the process of ultraviolet-induced DNA excision repair

The effect of 3-aminobenzamide, a potent inhibitor of poly(ADP-ribosyl)ation, on UV-induced DNA excision repair was investigated. HeLa cells were treated with DNA replication inhibitors, hydroxyurea (HU) and 1-beta-D-arabinofuranosyl cytosine (araCyt), before and after ultraviolet light (UV) irradiation, to accumulate DNA single-strand breaks. The activity of poly(ADP-ribosyl)ation measured in the permeable cell system of HeLa cells was enhanced in a UV dose-dependent manner after the combined treatment with HU and araCyt in vivo. However, DNA repair synthesis in vitro was not affected by addition of 1 mM 3-aminobenzamide or nicotinamide, while incorporation of [3H]NAD in the same system was completely inhibited. Furthermore, neither the magnitude of UV-induced DNA single-strand breaks accumulated by the combined treatment of HU and araCyt nor the rate of their rejoining after release from the HU and araCyt block were influenced even in the presence of 10 mM 3-aminobenzamide. As the cytotoxicity of UV irradiation was significantly potentiated by 5 mM 3-aminobenzamide, these results suggest that poly(ADP-ribosyl)ation is involved in a process other than DNA excision repair induced by UV irradiation.     

DNA nucleotide excision-repair synthesis is dependent of pertubations of deoxynucleoside triphosphate pool size

We have investigated the effects of fluctuations in deoxynucleoside triphosphate (dNTP) pool size on DNA repair and, conversely, the effect of DNA repair on dNTP pool size. In confluent normal human skin fibroblasts, dNTP pool size was quantitated by the formation of [³H]TTP from [³H]thymidine; DNA repair was examined by repair replication in cultures irradiated with UV light. As defined by HPLC analysis, the [³H]TTP pool was formed within 30 min of the addition of [³H]thymidine and remained relatively constant for the next 6 h. Addition of 2–10 mM hydroxyurea (HU) caused a gradual 2–4-fold increase in the [³H]TTP pool as HU inhibited DNA synthesis but not TTP production. No difference was seen between the [³H]TTP pool size in cells exposed to 20 M/m² and unrradiated controls, although DNA-repair synthesis was readily quantitated in the former. This result was observed even though the repair replication protocol caused an 8–10-fold reduction in the size of the [³H]TTP pool relative to the initial studies. In the UV excision-repair studies the precense of hydroxyurea did not alter the specific activity of [³H] thymidine 5'-monophospahte incorporated into parental DNA due to repaier replication. These results suggest that fluctuations in the deoxynucleoside triphosphate pools do not limit the extent of excision-repair sythesis in human cells and demonstrate that DNA nucleotide excision-repair synthesis does not significantly diminish the size of the [³H]TTP pool.     

Unscheduled DNA synthesis in rat pleural mesothelial cells treated with mineral fibres

Unscheduled DNA synthesis (UDS) was studied in confluent rat pleural mesothelial cells (RPMCs) arrested in G0/G1 with hydroxyurea (HU) and treated with various fibre types, i.e., chrysotile, crocidolite or attapulgite. In addition, the effects of UV light and of benzo[a]pyrene were determined as references. Using autoradiography after [3H]thymidine incorporation ([3H]dThd), RPMCs treated with 4 micrograms/cm2 of chrysotile fibres exhibited a low but significant enhancement of net grains compared to untreated cells. Treatment with higher doses of chrysotile was not possible because of the impairment of microscopic observation due to the presence of the fibres. Using liquid scintillation counting, RPMCs treated with chrysotile or crocidolite showed a significant dose-dependent increase in [3H]dThd incorporation compared to untreated cells. In contrast, attapulgite did not enhance [3H]dThd incorporation compared to untreated cells. Treatment of RPMCs with 1, 2 or 4 micrograms/ml of benzo[a]pyrene resulted in a significant increase in [3H]dThd incorporation. In order to discount a possible role of S cells in the augmentation of [3H]dThd incorporation, despite the presence of 5 mM HU, S cells were counted by autoradiography. Results indicated that the percentage of S cells was similar in asbestos-treated and untreated cultures. Stimulation of the S phase also seems unlikely because treatment of RPMCs with asbestos fibres in the absence of HU resulted in a reduction of [3H]dThd incorporation attributed to an impairment of the S phase by the fibres. 1-4 micrograms/ml benzo[a]pyrene or 10-50 J/m2 UV light resulted in an approximate doubling of [3H]dThd incorporation. The effects of inhibitors of DNA repair were determined in chrysotile-treated RPMCs. [3H]dThd incorporation was inhibited by cytosine arabinoside and nalidixic acid. These results show that asbestos produces UDS in RPMCs.     

Genotoxicity of epoxy resin hardeners in the hepatocyte primary culture/DNA repair test

The genotoxicity of 9 chemicals used as epoxy resin hardeners was examined in the DNA repair test with rat hepatocytes. DNA repair synthesis was elicited by 7 chemicals, i.e., 4-aminodiphenyl ether, 4,4-diaminodiphenyl ether, 3,4,4′-triaminodiphenyl ether, 3,3′-dichloro-4,4′-diaminodiphenyl ether, 1,3-phenylenedi-4-aminophenyl ether, 4,4′-diaminodiphenyl methane and 4,4′-methylene-bis(2-chloroaniline).The positive results obtained with 4 epoxy resin hardeners of unknown carcinogenicity, i.e., 4-aminodiphenyl ether, 3,4,4′-triaminodiphenyl ether, 3,3′-dichloro-4,4′-diaminodiphenyl ether and 1,3-phenylene-di-4-aminophenyl ether suggest that they may be carcinogens. The genotoxicity of 1,4-phenylene-di-4-aminophenyl ether, of unknown carcinogenicity, and 4,4′-diaminodiphenyl sulfone, for which there is no sound proof of carcinogenicity, was not confirmed in the DNA repair test. The result with 4,4′-diaminodiphenyl sulfone was in agreement with its lack of mutagenicity in Salmonella typhimurium.     

Sensitization of rad mutants of Dictyostelium discoideum to ultraviolet light by postirradiation treatment with caffeine

The capacity of normal human cells to regulate DNA-repair pathways was examined. Synchronous populations of WI-38 human diploid fibroblasts were used to determine whether base-excision repair was increased as a function of the cell cycle. 2 parameters of the base-excision repair pathway were examined: (1) The induction of the DNA-repair enzyme uracil DNA glycosylase which functions in an initial step in base excision repair: (2) cell-mediated base-excision repair as measured by unscheduled DNA synthesis after exposure to sodium bisulfite or to methyl methanesulfonate. The glycosylase activity was increased 5-fold during cell proliferation; unscheduled DNA synthesis was enhanced 4- to 30-fold in a similar fashion. Equivalent results were observed where repair replication was quantitated using density-gradient analysis in the absence of hydroxyurea. The increase of the activity of the uracil DNA glycosylase and the enhancement of DNA repair occurred prior to the induction of DNA replication. Furthermore, at the maximal stimulation of DNA replication both glycosylase activity and DNA repair had substantially diminished. As the cells entered the second cell cycle, the glycosylase activity was again increased and then was again diminished. These results suggest that human cells actively modulate this DNA-repair pathway. The temporal stimulation of base-excision repair suggests the possibility that a DNA-repair complex may be formed prior to DNA replication to prescreen DNA and thus ensure the transfer of the correct genetic information to daughter cells.     

The study of DNA-repair defects using [125I]iododeoxycytidine incorporation as an assay for the growth of herpes simplex virus

[125I]Iododeoxycytidine incorporation was used to measure herpes virus (HSV-1) DNA synthesis following specific DNA damage. Xeroderma pigmentosum fibroblasts were less able to replicate UV-irradiated viral DNA than were normal fibroblasts, indicating the necessity for excision repair for the survival of UV-irradiated virus. Because of its rapidity and ease of quantitation, this assay had advantages over standard viral mediated assays of DNA excision repair. It was possible to monitor viral replication as a function of the cellular cell cycle. Other genetic defects which have been proposed to reflect deficiencies in DNA-repair capacity were not detected by this assay. DNA-repair inhibitors, caffeine and 3-aminobenzamide, also did not show synergistic lethal effects on the replication of damaged viral DNA.