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Abstract: Sensitivity and correlations among three endpoints were evaluated to assess the genotoxic potential of organic complex mixtures in vitro. This study was focused on DNA adduct formation, DNA single strand break induction and tumour suppressor p53 protein up-regulation produced by extractable organic matter (EOM) absorbed on respirable particulate matter PM10 (particulate matter<10μm) collected in three European cities (Prague, Sofia, Košice) during winter and summer period. To compare the sensitivity of particular endpoints for in vitro measurement of complex mixture genotoxicity, the metabolically competent human hepatoma cell line Hep G2 was treated with equivalent EOM concentration of 50μg/ml. Cell exposure to EOMs resulted in significant DNA adduct formation and DNA strand break induction, however, a lack of protein p53 up-regulation over the steady-state level was found. While the maximum of DNA strand breaks was determined after 2h cell exposure to EOMs, 24h treatment interval was optimal for DNA adduct determination. No substantial location- and season-related differences in EOM genotoxicity were detected using DNA strand break assessment. In agreement with these results no significant variation in DNA adduct levels were found in relation to the locality and season except for the monitoring site in Prague. The Prague EOM sample collected during summer period produced nearly three-fold lower DNA adduct level in comparison to the winter EOM sample. Comparable results were obtained when the ambient air genotoxicity, based on the concentration of carcinogenic PAHs in cubic meter of air (ng c-PAHs/m3), was elicited using either DNA adduct or strand break determination. In general, at least six-fold higher genotoxicity of the winter air in comparison to the summer air was estimated by each particular endpoint. Moreover, the genotoxic potential of winter air revealed by DNA adduct assessment and DNA strand break measurement increased in the same order: Košice≪Prague

5-Fluorouracil (5-FU) is an essential chemotherapeutic drug for colorectal cancer (CRC) treatment. However, the frequent development of drug resistance has dramatically affected its clinical use. Therefore, novel treatment strategies are critical to improving patient outcomes. Herein, we investigated the ability of the epigenetic drug SAHA to increase the sensitivity of chemoresistant CRC cells to 5-FU. In addition, we evaluated the potential genotoxic risk of SAHA+5-FU combination treatment. As a model system, we used three CRC cell lines, HT-29, SW480, and HT-29/EGFP/FUR, differing in their resistance to 5-FU. CRC cell lines were exposed to sub-toxic SAHA concentrations for 24 h, followed by a 48 h treatment with 5-FU. The cytotoxicity of SAHA, 5-FU, and SAHA+5-FU was measured by the MTT test, the genotoxicity by the comet assay, and the micronucleus test. The apoptotic/necrotic activity was assessed using morphological criteria. We found a synergic decrease in the viability of HT-29 and SW480 cells, but not the most resistant HT-29/EGFP/FUR cells after combined SAHA+5-FU exposure compared to 5-FU. Remarkably, SAHA most efficiently induced apoptosis in HT-29/EGFP/FUR cells compared to HT-29 and SW480 cells. Combined SAHA+5-FU treatment resulted in a synergistic increase in apoptotic/necrotic cells in HT-29 cell line, while rather additive/sub-additive effect was determined in the SW480 and HT-29/EGFP/FUR cells. At the same time, however, a synergistic rise in micronuclei was found in CRC cell lines (at least at some concentrations). We have shown that SAHA can sensitize CRC cells to 5-FU; therefore, epigenetic and convential drug combinations could be beneficial for the patients. However, the increase in micronucleus formation after combined SAHA+5-FU treatment indicates a potential health hazard. The clastogenic activity could contribute to cancer heterogeneity, favoring progeny of such aberrant cells to clonal expansion. Therefore, developing new specific epigenetic drugs or nanocarriers for targeted drug delivery might reduce the potential genotoxic risk., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The aim of this study was to evaluate radiosensitivity of cervical cancer cells in vitro and to assess the relationship between genetic polymorphisms in DNA repair genes and the response of cells to ionizing radiation. The alkaline comet assay as a predictive assay of radiosensitivity was used to examine the susceptibility of four human cervical cancer cell lines (CaSki, C-33A, HeLa and SiHa) to radiation damages. The initial DNA damage and the residual DNA damage at 15, 30, 45 and 60 min after irradiation were assessed. Genotypes of DNA repair genes (XRCC1, hOGG1, PARP, XPD, XRCC3 and XRCC4) were analyzed by PCR-RFLP assays. The comet data clearly indicate a variable but dose-dependent increase in the initial DNA damage in all cell lines. The highest slope of dose response curve was observed in C-33A cells and this cell line was assumed to be radiosensitive. All cell lines repaired DNA damage in a similar manner, the level of DNA strand breakage has returned near the background level within 45 min after irradiation. According to the genotype we found that C-33A cells are polymorphic in the majority of analyzed DNA repair genes. This pilot study indicated associations between polymorphisms in DNA repair genes and cell radiosensitivity.

Post-cultivation of treated cells in the presence of DNA repair inhibitors has been proposed as a new methodological approach of the micronucleus (MN) assay to increase the sensitivity of this technique. In order to assess the advantages and limitations of this promising methodological approach, several genotoxic/clastogenic agents with different mechanisms of activity were chosen to assess the effect of DNA repair inhibitors on the level of micronuclei (MNi) induced by particular agent using Chinese hamster V79 cells. Both UV light (UV) and benzo(a)pyrene (BaP) increased significantly the micronucleus level in V79 cells (p<0.01-0.001). In contrast, only at cytotoxic concentration (>0.8 mM) a slight but statistically significant rise of MNi was determined in cells exposed to N-methyl-N-nitroso urea (MNU). However, post-cultivation of MNU-treated cells in the presence of DNA repair inhibitors (cytosine arabinoside, AraC and hydroxyurea, HU) led to an additional rise of MNi. While AraC had a synergistic effect on MN formation (0.4 mM and 0.8 mM, DS=2.14 and 1.13, respectively), HU had less than additive effect (DS=0.86 and 0.66) and the combined treatment of cells with AraC and HU was least effective (Cf=0.36 and 0.28). On the other hand, post-cultivation of UV- and BaP-treated cells in the presence of AraC did not result in any synergistic effect on MN formation. No effect or even a decrease of MNi was measured particularly due to HU or combined treatment of HU and AraC. Incubation of control untreated cells with AraC gave rise to a significant increase of MN formation (2- to 2.5-fold) as well. Hydroxyurea or the combined treatment of HU with AraC had lower effect on the spontaneous level of MNi. Our study shows, that the combination of MNU treatment with DNA repair inhibitors increased the number of MNi on well proliferating V79 cells; in case UV light and BaP treatment, the involvement of DNA repair inhibitors did not contribute to an increase of sensitivity of MN assay. On the basis of our results we suppose that the AraC/CBMN assay might be a promising approach in genetic toxicology applied only to lymphocytes.

Hepatocytes freshly isolated from male Wistar rats fed a common diet or a vitamin A- or vitamin E-supplemented diet (each for 21, 28, or 41 days) were assayed for sensitivity to DNA breakage and cytogenetic changes induced by carcinogens. Different indirectly acting carcinogens were assayed. N-nitrosomorpholine (NMOR) was the only agent that induced DNA breaks, chromosomal aberrations, and micronuclei in all experiments. Benzo[a]pyrene (B[a]p) and dimethyldibenzo [c,g]carbazole (diMeDBC) induced only DNA breaks in all experiments. Occasionally, B[a]P induced chromosomal aberrations and micronuclei, and diMeDBC induced micronuclei, but not chromosomal aberrations. These results demonstrated that the tested carcinogens assayed at concentrations highly effective in a hypoxanthine phosphoribosyltransferase/V79 system significantly increased DNA damage, while cytogenetic changes were less frequent. In hepatocytes from rats fed vitamin A, a reduction in the severity of all three end points was observed after NMOR treatment. After B[a]P treatment, we found a reduction in DNA breaks and chromosomal aberrations; after treatment with diMeDBC, we observed a reduction in DNA breaks. Treatment with vitamin E was less effective: it reduced DNA strand breaks induced by B[a]P and partially reduced those induced by diMeDBC and NMOR and the level of micronuclei induced by NMOR and B[a]P. Both vitamins reduced the level of DNA strand breaks induced by the oxidative effect of a visible light-excited photosensitizer.

The ability of carboxymethylglucan (CMG), a high molecular water-soluble derivative of glucan, was evaluated to act as a scavenger of reactive oxygen species. Hydrogen peroxide and methylene blue plus visible light, well-defined oxidant factors, were used as a model agents for induction ofoxidative DNA damage in CaCo-2 cells. Both hydrogen peroxide and visible light gave rise to dose-dependent increase of DNA damage mediated by hydroxyl radicals (*OH) or singlet oxygen (1O2), respectively. While DNA lesions generated by hydrogen peroxide dominated by strand breakage, exposure of CaCo-2 cells to visible light led mainly to base modifications sensitive to formamidopyrimidine DNA-glycosylase (Fpg). Neither CMG nor ascorbic acid, a known antioxidant, induced any DNA damage in CaCo-2 cells. Pretreatment of cells with ascorbic acid prior to H2O2 or visible light exposure resulted into statistically significant reduction of DNA lesions induced by particular agent. However, pretreatment of CaCo-2 cells with CMG in concentration range from 0.01 microM to 1 microM reduced neither the level of strand breaks induced by hydrogen peroxide nor the number of Fpg-sensitive base modifications generated by visible light.

Radiation-induced DNA damage and kinetics of DNA repair was evaluated in three human ovarian carcinoma cell lines (i.e. CH-1, A-2780 and SKOV-3) with different sensitivities to ionizing radiation and radiation-induced apoptosis with the aid of single cell gel electrophoresis (SCGE, the comet assay). A good correlation was found between the initial level of DNA breaks and radiation induced apoptosis in CH-1 and SKOV-3 cell lines. While the radiation-sensitive CH-1 cell line manifested the highest level of initial DNA breakage and a significant delay in DNA break rejoining, the inverse correlation was found in the radiation-resistant cell line SKOV-3. Intermediate initial level of breaks was induced in the A-2780 cell line characterized by the intermediate sensitivity to X-ray radiation in comparison to CH-1 and SKOV-3 cells, however, the kinetics of DNA repair was comparable with radiation-resistant cell line SKOV-3. Our data suggest that the comet assay could be a promising tool for prediction of intrinsic cell radiosensitivity. This method might be considered as a supplementary technique to the more reliable but time consuming clonogenic assay.

Single cell gel electrophoresis (SCGE) or comet assay is a rapid and sensitive fluorescent microscopic method which allows measurement of DNA strand breaks in individual cells. Modifications of SCGE conditions permitted to detect different types of DNA damage. In order to characterize DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and hydrogen peroxide (H2O2) in Chinese hamster V79 cells, two approaches were used: (1) two pH values of unwinding and electrophoresis solutions (pH > or = 13.0 and pH = 12.1) to specify the type of DNA lesions [the alkali-labile sites and true DNA single-strand breaks (ssb)] and (2) DNA glycosylases [endonuclease III (EndoIII) and formamidopyrimidine-DNA glycosylase (FaPy)] or DNA inhibitors [hydroxyurea (HU) + 1-(beta-D-arabinofuranosyl)cytosine (AraC)] to characterize the types of DNA damage. Our results showed that the lesions induced by H2O2 represented mainly the true DNA ssb, while MNNG formed predominantly alkali-labile sites, which were converted to DNA ssb under strong alkaline conditions (pH > or = 13.0). The effects of DNA repair enzymes and DNA inhibitors were more significant under lower pH (pH = 12.1) of unwinding and electrophoresis solution. Both, DNA glycosylases and DNA inhibitors increased the level of DNA ssb.

Single cell gel electrophoresis (SCGE) was used to evaluate the level of DNA damage in peripheral blood (PB), bone marrow (BM), and lymphatic node (LN) cells of patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML) and non-Hodgkin's lymphoma (NHL). The level of DNA damage was compared with the level of basal DNA damage in control group, represented by healthy donors. Statistically significant increase of basal DNA damage was found in leukemia/lymphoma cells of patients suffered from AML, CML, ALL of T-cell subtype (T-ALL), and NHL, however, no difference in basal DNA damage was found in patients with ALL of early B-cell subtype (B-ALL) and CLL in comparison to control group. The mean basal DNA damage increased in the order CLL

Three techniques: single cell gel electrophoresis (SCGE), alkaline elution of DNA (AE), and alkaline DNA unwinding (ADU) were chosen to compare the sensitivity among these methods in detection of DNA damage and repair in human diploid VH10 cell line after short-term exposure to hydrogen peroxide. Using SCGE technique a dose-dependent increase in DNA migration was found in cells exposed to hydrogen peroxide in concentration range from 10 micromol/l to 100 micromol/l. Alkaline DNA unwinding method detected increased level of single strand breaks (ssb) in concentration range from 25 micromol/l to 100 micromol/l of H2O2, and alkaline elution of DNA estimated increased DNA elution rate from concentration 50 micromol/l of H2O2. In a time course study to evaluate the kinetics of DNA repair, both SCGE and ADU techniques showed that the repair of DNA strand breaks is very rapid; the level of ssb in treated cells has returned to near the background level within two hours. After this time damage remaining in the DNA was in the form of oxidised bases as revealed the incubation of treated cells with specific DNA repair endonuclease, formamidopyrimidine-DNA glycosylase.

We describe an alternative assay to determine genotoxicity. Its main feature is that it combines two measures in a single experiment; the inhibition of replicative DNA synthesis together with the stimulation of DNA repair. We show that, in tests of four different genotoxic agents, the assay gives results that are entirely consistent with what is known about the mode of action of these agents. In addition, we have demonstrated that chemical carcinogens requiring metabolic activation can be examined using a standard procedure of incubation with a microsomal activating fraction. We consider the combined assay for DNA synthesis inhibition and repair synthesis to be a useful way for the rapid prescreening of chemicals suspected of genotoxic activity on the level of mammalian cells.

In a study of potential carcinogenicity of pesticides, Piritione, metolachlor (in the form of Dual and VUCHT 524) and Supercypermethrin (in the form of Supercypermethrin EC and Supercypermethrin TP) were assayed for induction of anchorage independent growth of BHK21 cells and morphological transformation of Syrian hamster embryo cells. The activity of these substances in both transformation assays was compared to the activity of the direct-acting ultimate carcinogen N-methyl-N-nitrosourea. In comparison to the very strong transforming activity of N-methyl-N-nitrosourea all pesticides tested with or without S9 fraction manifested a very weak, weak, medium or strong effect. The ability to induce anchorage independent growth was graded as follows: Dual < Supercypermethrin EC < Supercypermethrin TP < or = Piritione < VUCHT 524. Results of Syrian hamster embryo cell transformation assay were very similar to the BKH21 transformation assay. VUCHT 524 strongly induced transformation whereas Dual was inactive. Piritione and Supecypermethrin EC and Supercypermethrin TP elicited a slight but significant positive response.

Many chemicals which promote tumorigenesis in vivo have been observed to inhibit metabolic cooperation between 6-thioguanine-resistant (6TGr) and sensitive (6TGs) Chinese hamster lung V79 cells. The apparent correlation between inhibition of metabolic cooperation in V79 cells in vitro and promotion of oncogenesis in vivo has led to the suggested utilization of the assay as a screen for tumor promoters. Several parameters concerning the V79 metabolic cooperation assay were investigated for an improved understanding of the usefulness and limitations of the assay in our laboratory conditions. We have found that the recovery of 6TGr cells were dependent upon the number of 6TGs cells plated, upon the generation time, passage number, and upon the co-cultivation period until the addition of selective agent. The ability of this assay to detect tumor promoters has been determined by the known tumor promoter phorbol ester TPA.

After treatment with methyl methanesulfonate (MMS) or N-methyl-N-nitrosourea (MNU), the mutagenicity and survival of Chinese hamster V79 cells were investigated, as well as the inhibition of daughter DNA synthesis and, using the DNA unwinding technique and hydroxylapatite chromatography, the character of the newly synthesized DNA was studied. It was found that different cytotoxicity and mutagenicity of MMS and MNU was accompanied by different types of DNA synthesis inhibition. The treatment with the former compound resulted in a longer inhibition of DNA synthesis, while the treatment with the latter showed that as early as 2 h after exposure the percentage of nascent DNA increased. Shortly after the exposure to both alkylating agents, the newly synthesized DNA contained a higher number of gaps than control DNA, in dependence on the concentration used. During culturing after treatment, the character of nascent DNA in MMS-treated cells gradually returned to that of control DNA, while MNU-treated cells, for the whole time of our study, synthesized DNA with a larger number of gaps than control DNA. We suggest that the character of nascent daughter DNA reflects the occurrence of lesions in parental DNA. These are repaired within a shorter time in MMS- than in MNU-treated cells. The long-term persistence of lesions in the DNA of MNU-treated cells might be one of the factors responsible not only for the higher cytotoxic but also for the many times higher mutagenic effect of this alkylating agent.

The ability to induce gene mutations at the HGPRT locus in Chinese hamster V79 cells and to inhibit DNA synthesis in human EUE cells has been followed in eighteen chemical substances and ultraviolet light. The aim was to determine whether following of inhibition of DNA synthesis in impaired human cells may serve as an indicator of the mutagenicity of the relevant chemical substance. The results permit us to assume that following of DNA synthesis, the so-called DNA-inhibition test provides a preliminary information on the genotoxicity of the chemical substance, although the degree of inhibition of DNA synthesis need not necessarily denote the measure of the mutagenic effect of the substance. As certain further tests, utilising in vitro mammalian cells, so also the DNA-inhibition test yields a certain percentage of false positive results. Hence, it is important to combine this test with further express in vitro tests on the level of mammalian cells.