Chromosomal changes induced by chrysotile fibres or benzo-3,4-pyrene in rat pleural mesothelial cells

The induction of chromosomal aberrations in rat pleural mesothelial cells (RPMC) following in vitro treatment with chrysotile fibres has been demonstrated. The production of chromosomal aberrations was also observed after treatment of the cells with benzo-3,4-pyrene (BP). The yield of abnormal metaphases was dose-dependent and reached 58% at a BP dose of 2 micrograms/ml. Chrysotile fibres at 7 micrograms/ml induced 21% abnormal metaphases and the frequency decreased with further increases in fibre concentration. Their decline is possibly related to a lethal effect. Chrysotile-induced chromosomal aberrations were primarily of the chromatid type and included breaks and fragments. BP induced chromosome exchanges which were not seen following chrysotile treatment. Minutes and double minutes were detected in BP-treated RPMC and occasionally found after chrysotile application. These results confirm that chrysotile fibres are clastogenic for some cultured cells and demonstrate that the fibres induce chromosome damage in target RPMC.     

Unscheduled DNA synthesis of rat hepatocytes in monolayer culture

Monolayer cultures of rat hepatocytes activated tris(2,3-dibromopropyl)phosphate (Tris-BP) more efficiently than 2-acetylaminofluorene (AAF), to genotoxic products which caused mutations in co-cultures of S. typhimurium. In contrast, AAF caused a greater genotoxic response in the hepatocytes than Tris-BP, as judged by the increase in DNA-repair synthesis measured by liquid scintillation counting of ³H-TdR incorporated into DNA isolated from the nuclei of the hepatocytes. Covalent binding of 0.05 mM ³H-Tris-BP to cellular proteins occurred at a similar rate as covalent binding of 0.25 mM ¹⁴C-AAF. Tris-BP was the more cytotoxic of the two compounds as determined by leakage of cellular lactate dehydrogenase into the culture medium. The observed differences in the cytotoxic and genotoxic responses between Tris-BP and AAF were probably caused by differences in the nature of their reactive metabolites with respect to stability, lipophilicity and/or their interactions with variuos cellular nucleophilic sites. The relative DNA-repair synthesis induced by an AAF exposure for 18 h decreased with time after plating of isolated hepatocytes. Tris-BP first caused an increase in the relative DNA-repair synthesis up to 27 h after plating, whereafter the response declined reaching control values using cultures 75 h after plating. In parallel with the decreased relative response in DNA-repair synthesis with time, the background radioactivity in isolated nuclei from untreated cells increased both when the hepatocytes were incubated in the presence or absence of hydroxyurea to inhibit replicative DNA synthesis. Increased DNA-repair synthesis was demonstrated as early as 3 h after commencing exposure to the test substances. While the induced DNA-repair synthesis caused by Tris-BP remained constant after 6 h of exposure, the response caused by AAF increased with increased exposure time beyond 6 h. To assess the role of different metabolic pathways in the genotoxic and cytotoxic responses of Tris-BP and AAF, the hepatocytes were exposed to test substances in the presence of various metabolic inhibitors for 3 h, whereafter the cell medium was removed and replaced by cell-culture medium containing ³H-TdR and hydroxyurea. The cytochrome P-450 inhibitor metyrapone decreased both the genotoxic and cytotoxic effects of Tris-BP, while α-naphthoflavone reduced the genotoxic effect of AAF. The addition of glutathione (GSH) or N-acetylcysteine decreased both the cytotoxic and genotoxic effects of Tris-BP, while cellular depletion of GSH by diethylmaleate increased these effects. Manipulations in the cellular levels of sulhydryl-containing substances in the hepatocytes by these agents had little effects on the DNA-repair synthesis caused by AAF. The results indicate that such a hepatocyte culture system may be very useful as a tool to study mechanisms involved in the formation of cytotoxic and/or genotoxic metabolites from various xenobiotics.     

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.     

Rat pleural mesothelial cells in culture

Summary A culture system has been developed for long-term maintenance of rat pleural mesothelial cells. Mesothelial cells were isolated from the parietal pleura of rats and cultured in NCTC 109 medium supplemented with 10% fetal bovine serum. The cell explants attached to the dish and formed a confluent monolayer of polygonal cells within 10 to 15 days. Subcultures were made in the same medium. The mean population doubling time was approximately 30 hr. The ultrastructure of the mesothelial cells in culture was studied by light and electron microscopy and was compared with that of cells obtained from submesothelial components. This work was supported by CEE Grant No. 264 77 6 ENV F.     

Unscheduled DNA synthesis in human leucocytes

Polymorphonuclear leucocytes have been induced to synthesize new DNA by exposure to UV light. Preliminary observations (not included) also indicate that 6-MeV electrons and incubation with the radiomimetic agent methyl methanesulfonate (MMS) are effective agents for inducing unscheduled DNA synthesis (UDS). A study of the kinetics of UV-induced DNA synthesis suggests that there are at least two processes operating, one fast and essentially complete within the first 1–2 h and the second lasting at least 8 h.     

Unscheduled DNA synthesis in spermatogenic cells of mice treated in vivo with the indirect alkylating agents cyclophosphamide and mitomen

Cyclophosphamide (CPA) and mitomen (DMO) are chemical mutagens that require metabolic activation to produce their biological effect. We have used an in vivo UDS assay in various meiotic and postmeiotic germ-cell stages of male mice to study DNA repair after treatment with these chemicals. EMS, a compound requiring no metabolic activation, was also used for comparative purposes.CPA and DMO induced UDS in meiotic through early-to-midspermatid stages, but no UDS was detected in late spermatids and mature sperm. While EMS produced a maximum UDS response in the germ cells immediately after treatment, CPA and DMO did not produce a maximum response until ～0.5 to 1 h after injection. This delay is attributed to the time required for CPA and DMO to be enzymatically vonverted active alkylating metabolites.Unlike the results found with EMS, mutation frequencies (dominant lethals, translocations, specific-locus mutations) following CPA treatment are not noticeably reduced in germ-cell stages in which UDS occurred. In the case of DMO, mutations are induced only in mature spermatozoa, and these germ-cell stages represent only a fraction of those in which no UDS is detected. The results with CPA and DMO thus still leave unclear the relationship between DNA repair and the differential spermatogenic response of mice to genetic damage.     

Growth of normal and neoplastic rat pleural mesothelial cells in the presence of conditioned medium from neoplastic mesothelial cells

Several transformed cells have been demonstrated to secrete growth factors. We studied the effect of conditioned medium from neoplastic rat pleural mesothelial cells on normal and neoplastic mesothelial cell growth. The results showed that the concentrated conditioned medium stimulated neoplastic mesothelial cell growth but inhibited reversibly normal mesothelial cell growth.     

Occurrence and morphology of tumors induced in nude mice transplanted with chrysotile-transformed rat pleural mesothelial cells

Rat pleural mesothelial cells treated in vitro with chrysotile fibers have been successfully transplanted into nude mice. Three cultures (1 untreated, 2 treated) were injected at passage 75; a fourth culture was obtained from a mesothelioma induced in rat by chrysotile fibers. Overall, tumors grew in each series, but the delay between cell injection and tumor formation was 22 wk with untreated cells whereas only 1 or 2 wk were needed with treated cells, and 1 wk with cells from in vivo-induced mesothelioma. Pathological study by light and electron microscopy of tumors is reported here and showed the mesothelial nature of the cells. Comparison between the ultrastructure of the injected cells and tumor cells indicated that the morphology of injected cells was retained in tumors even if the delay in tumor formation was long. These results suggest that this model is useful for investigating mesothelial cell transformation resulting from in vitro or in vivo exposure to certain carcinogens.     

Metabolism of benzo(a)pyrene in rat pleural mesothelial cells in culture

Cultures of Rat pleural mesothelial cells obtained after isolation of parietal cells metabolized benzo(a)pyrene to organosoluble and hydrosoluble derivatives. This property is well maintained since it was observed with cells at 6th passage as well as after 39 passages.     

Unscheduled DNA synthesis: Some statistical thoughts

Statistical interpretation of results of experiments involving unscheduled DNA synthesis is examined from a design standpoint. Most appropriate methods currently in use are evaluated and some modifications and extensions are suggested. Concerns about replication and/or interaction errors are evaluated and methods for their appropriate handling are discussed. It is suggested that methods incorporating both dose-response and heterogeneity statistics should be considered in treating results from unscheduled DNA synthesis experiments. Proper designs for such experiments are emphasized.Abbreviations ANOVA analysis of variance - MSE mean square error - UDS unscheduled DNA synthesis     

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.     

Unscheduled DNA synthesis in xeroderman pigmentosum cells after microinjection of yeast photoreactivity enzyme

Photoreactivating enzyme (PRE) from yeast causes a light-dependent reduction of UV-induced unscheduled DNA synthesis (UDS) when injected into the cytoplasm of repair-proficieint human fibroblasts (Zwetsloot et al., 1985). This result indicates that the exogenous PRE monomerizers UV-induced dimers in these cells competing with the endogenous excision repair. In this paper we present the results of the injection of yeast PRE on (residual) UDS in fibroblasts from different excision-deficient XP-strains representing complementation groups A, C, D, E, F, H and I (all displaying more than 10% of the UDS of wild-type cells) and in fibroblasts from two excision-proficient XP-variant strains.In fibroblasts belonging to complementation groups C, F and I and in fibroblasts from the XP-variant strains UDS was significantly reduced, indicating that pyrimidine dimers in these cells are accessible to and can be monomerized by the injected yeast PRE. The UDS reduction in the XP-variant strains is comparable with the effect in wild-type cells. In cells from complementation groups C, F and I the reduction is less than in wild-type and XP-variant cells. Fibroblasts belonging to groups A, D, E and H did not show any reduction in UDS level after PRE injection and illumination with photoreactivating light. These result give evidence that the genetic repair defect in some XP-strains is probably due to an altered accessibility of the UV-damaged sites.